Structure and Stability of Hyperstable Proteins: Glycolytic Enzymes From Hyperthermophilic Bacterium Thermotoga Maritima

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS: GLYCOLYTIC ENZYMES FROM HYPERTHERMOPHILIC BACTERIUM THERMOTOGA MARlTlMA By R. JAENICKE, H. SCHURIG, N. BEAUCAMP, and R. OSTENDORP lnstltut far Blophyslk und Physlkallsche Blochemle, Unlversltht Regenaburg, 0-93040 Regensburg, Germany

1. Introduction ...................................................... 11. Fundamentals of Protein Stability .............................. A. Thermodynamic Aspects . . . . .............................. B. Forces Involved in Protein Stabilization .......................... C. Structural Increments of the Free Energy of Stabilization .......... D. What Model Studies Teach about Stabili ......................

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A. Limits of Growth versus Limits of Thermal Stability: Model Studies . B. Proteins from Hyperthermophilic Organisms ..................... TV. Hyperthermophilic Bacterium T h a o t o g u mun’tzmu .................... V. Glycolytic Enzymes . . . . . . . . . . ............ A. Triose-Phosphate Isomerase .................................... B. Glyceraldehyde-%PhosphateDehydrogenase ...................... C. Phosphoglycerate Kinase and Phosphoglycerate Kinase-Triose-Phosphate Isomerase Fusion Protein ...................................... D. Enolase ...................................................... E. Lactate Dehydrogenase ........................................ VI. Conclusions ..... ................................... References .......................................................

I.

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INTRODUCTION

Proteins exhibit marginal stabilities, equivalent to only a few weak intermolecular interactions. Extreme conditions require either molecular adaptation in terms of local structural changes or stabilization by “extrinsic factors” not encoded in the amino acid sequence. Such factors include compatible solutes, specific ions, metabolites, cofactors, etc. Under physiological stress, increased turnover may compensate for denaturation; molecular chaperones may also play an important role as salvage systems. N o general strategy of stabilization has yet been established. However, certain incremental contributions to stability have been elucidated by analyzing extremely stable proteins, for example, those from hyperthermophiles, on one hand, and stabilizing or destabilizing point mutants, on the other. Stabilization may involve all levels of the hierarchy ADVANCES IN PROTEIN CHEMISTRY. Vol. 48

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Copyrighl 0 1996 by Academic Press. Inc. All righu of reproduction in any form reserved.

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of protein structure, that is, local packing of the polypeptide chain in secondary or supersecondary structural elements, as well as domains and subunits. Experimental approaches that have been used to assign specific structural alterations to changes in stability are (1) selection for temperaturesensitive (ts) mutants, (2) systematic variations of amino acid residues in the core or in the periphery of model proteins, (3) fi-agmentation of domain proteins or alteration of connecting peptides hetween domains, and (4) alteration of subunit interactions by mutagenesis or solvent perturbation. Data have been summarized in a number of reviews (Privalov, 1979, 1982; Baldwin and Eisenberg, 1987; Matthews, 1987, 1991; Alber, 1989a,b; Privalov and Gill, 1989; Dill, 1990; Pace, 1990; Jaenicke, 1991a; Matthews, 1993). In this article, we follow a different approach, comparing selected proteins from a hyperthermophilic microorganism with corresponding homologs from mesophilic sources. Apart from the wild-type proteins and mutants with varying intrinsic stabilities, mechanisms of stabilization are discussed, based on model systems such as subdomains, domains, and subunits. It is obvious that the solution of the stability problem has biological and technological implications: biological because of the evolutionary aspects of adaptive response reactions to physiological stress, and technological because the molecular mechanisms of thermal adaptation might be applicable in producing engineered thermostable proteins in connection with bioreactors and biosensors. As indicated by the distribution of organisms, especially microorganisms, over the whole surface of the earth, there is an enormous adaptive capacity of life in the biosphere: centers of volcanic action seem to be the only areas where no traces of growth are found. The presently reported (hypothetical) upper temperature limit of viability is approximately 150°C (Stetter, 1992); at low temperature, cryptobiosis (i.e.,c r y p tic life still accessible to full recovery of all cellular functions) has been observed to temperatures as low as -40°C (I. E. Friedmann, personal communication 1995).In contrast to the extremes of pH arid low water activity, there is no way of avoiding thermal stress because microorganisms in their habitat are isothermal. Thus, in order to survive, they have to adapt their cell inventory to the respective temperature such that they are resistant against heat or cold denaturation. As has been mentioned, the protection may be accomplished by either intrinsic or extrinsic stabilization Uaenicke, 1991a). In most cases, adaptation in the course of evolution resulted in thermophily or psychrophily rather than t h e r m e tolerance. This means such organisms require either high or low temperature for their life cycle; they are unable to grow or multiply under

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mesophilic conditions. Widely accepted optimal growth temperatures are: 280°C for hyperthermophiles, 250°C for thermophiles, 225°C for mesophiles, and <25”C for psychrophiles. In this chapterwe focus mainly on a representative hyperthermophilic bacterium, Thermotoga maritima ( T. maritima, or Tm as prefix of enzymes),which has optimized its cellular functions to a maximum growth temperature of about 90°C. As shown by 16 S rRNA sequence analysis, hyperthermophiles are close to the root of the phylogenetic tree, suggesting that they preceded their mesophilic counterparts (Woese and Fox, 1977; Woese et al., 1990; Stetter, 1992, 1993). Considering the single adaptive steps in the process of evolution, this finding is significant because thermophilic adaptation would require the entire protein inventory of the cell to be stabilized in a synchronous fashion. With a (hyper-)thermophile as the universal ancestor, and the expansion of life from high to low temperatures, single temperature-sensitive mutations are sufficient to explain how life could “multiply and replenish the earth” to the extent that organisms spread practically over the whole surface of the planet. The strategies allowing full metabolic and reproductive activity under conditions close to the limits of thermolytic degradation of biomolecules are expected to be manifold. Available data from model systems such as T4 lysozyme (Matthews, 1987,1991)or P22 tailspike protein (Mitraki and King, 1992;Mitraki et al., 1993)do not allow general rules of thermal a d a p tation to be defined. One reason is that minute local structural alterations are sufficient to provide significant contributions to the free energy of stabilization.Obviously, there are many ways to accumulate the difference in free energy, AAG,,,, that is required to shift the optimum of growth or viability from mesophilic to thermophilic or even hyperthermophilic conditions. Another reason is the fact that environmental factors are interrelated by physicochemical laws, leading to indirect effects, apart from the direct ones, discussed so far. For example, temperature affects the solubility of solvent components, as well as the viscosity, ionization, and kinetics of chemical reactions. In vitroexperimentsmay be devised to separate these effects; however, evolution had to cope with the complex superpositions, rendering the analysis of specific strategies of adaptation in the intricate interplay of variables in the cell extremely difficult. The following sections illustrate this complexity. The examples given allow two general conclusions: first, thermophilic (and other extremophilic) proteins do not exhibit properties qualitatively different from nonextremophilic ones; second, the essential adaptive alterations tend to shift the normal characteristics to the extreme in the sense that under the respective physiological conditions the molecular properties are comparable. This way, adaptation of biomolecules to extremes of physical

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conditions tends to maintain “corresponding states” regarding overall topology, flexibility, and solvation. OF PROTEIN sTABI1.lTY 11. FUNDAMENTALS

The term protein stability refers to the preservation of the unique chemical and spatial structure of a polypeptide chain under extremes of physical conditions. Denaturation and stability are interconnected since perturbing the native structure of a protein is the only way to quanufy its stability. Ab initio calculations of the free energy of stabilization of proteins are not feasible. As we know from renaturation experiments, the functional state of globular proteins depends on their unperturbed three-dimensional structure (Jaenicke, 1987). The uniqueness of the functional state is stressed by two considerations: (i) high-resolution X-ray data for more than 800 crystal structures have clearly shown that the atomic coordinates of any group or residue in a crystallizablc globular protein can be determined with an accuracy of better than 2 A, or, after refinement, to better (ii) calculating all possible conformations of a polypeptide than 1 chain with an average size of a domain, the result amounts to the number of atoms in the universe (Dickerson and &is, 1969), and the native structure represents just one out of this astronomical quantity. To some extent, this description is misleading because the protein in its native threedimensional state is a dynamic system that may fluctuate between preferred conformations, as in the case of allosteric R and T states. The corresponding motions may be restricted to amino acid side chains, but they may equally well involve stretches of the polypeptide chain, or even domains and subunits. Their amplitudes and angles differ over wide ranges, with maximum values around 40 8, for chain and domain movements, and about 20” for domain rotations. The largest structural fluctuations and chain movements occur in processes such as enzyme activation and protein folding/unfolding. The biological significance of such motions becomes evident if one considers examples such as the uptake and release of oxygen by myoglobin, hinge-bending motions of antibody domains, or the coating of nucleic acid strands in viruses (Frauenfelder et al., 1979; Crumpton, 1986; Bennett and Huber, 1983;Jaenicke, 1987; Huber, 1988). Obviously, flexibility is a prerequisite of biological function, and any discussion of the structure-function relationship of proteins must include structural fluctuations of functional groups, chain segments, and domains. In connection with the stability of proteins from mesophiles compared to thermophilic homologs, it is important to note that the most signifi-

A;

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cant difference, for example, between glyceraldehyde-%phosphatedehydrogenase from yeast and T. mantima, refers to the drastic decrease in flexibility observed for the anomalously stable bacterial enzyme. Experimental evidence to prove this came from the following findings. The hydrogen-deuterium (H-D) exchange rates of the thermophilic enzyme at 25°C are only about 60% of those of the yeast enzyme. At room temperature, the Tm enzyme shows only marginal catalytic activity, but this can be dramatically enhanced by nondenaturing guanidine concentrations (Wrba et al., 1990a; Rehaber and Jaenicke, 1992). A.

l h m o d y n a m i c Aspects

As mentioned, protein stability is a cumulative effect of local interactions. These accumulate in the process of folding in the sense that secondary and supersecondary structural elements, subdomains, domains, and, finally, subunits coalesce and associate in a sequential manner. The initiation of the folding reaction is assumed to occur at specific nucleation points. Their stabilities determine the energetics and kinetics of protein self-organization. Since what is called the denatured state in fact represents an ensemble of readily interconvertible conformers with equal or closely similar energies, there is no way to unravel the detailed pathway of folding in terms of an unambiguous description of the sequence of events in the transition from the unfolded state (U), via intermediates ( I ) , to the native (N) state:

Structural intermediates of general significance that have been investigated in detail are (1) the molten globule state as an early collapsed state on the folding path, (2) molecules with wrong proline isomers, and (3) disulfide cross-linking intermediates on the folding pathway of small model proteins (Creighton, 1978, 1990;Jaenicke, 1991b; Christensen and Pain, 1991; Schmid, 1993; Schmid et al., 1993; Ptitsyn, 1992). Since N may be considered a well-defined, unique state, the reverse reaction of Eq. (1) is expected to provide insight into the unfolding reaction in terms of the sequential loosening of local interactions. Making use of H-D exchange kinetics of amide protons and nuclear magnetic resonance (NMR), Kiefhaber and Baldwin (1995) were able to show that a single rate-limiting step in unfolding breaks the entire network of hydrogen bonds and causes the overall unfolding of ribonuclease A. The detailed analysis of equilibrium unfolding intermediates proves that the antiparallel 0 strand represents the stable backbone of the molecule,

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whereas the helix exhibits lower stability (T. Kiefhaber, personal communication 1996). In referring to intermediates in the processes of folding/unfolding, it is important to explore whether thermal denaturation is a two-state process or whether intermediates represent a significant population in the equilibrium transition K

N

#

U.

The best criterion for two-state behavior is the comparison of the calorimetrically determined enthalpy, AHcd,with the corresponding van’t Hoff enthalpy (AHVH)calculated from the temperature dependence of the equilibrium constant K For a variety of single-chain proteins, the ratio AHc,/AHvH has been shown to deviate only slightly from unity, so that the concentration of intermediate states over the whole transition range is insignificant. Obviously, intermediates are thermodynamically highly unstable relative to the two macroscopic states, N and U. The high cooperativity of the transition suggests that N requires all its stabilizing interactions to maintain its functional state or, vice versa, all essential stabilizing elements responsible for the native conformation occur simultaneously at the final stage of folding (Privalov, 1979; 1982; Creighton, 1990). For domain proteins such as papain or immunoglobulin, large deviations of the AHCaI/AHvH ratio from unity have been observed, proving that there exist independent cooperative units. In certain cases their unfolding transitions are well separated so that intermediates may be highly populated (see Section 11,B). The most important thermodynamic parameter characterizing protein denaturation is the large positive change in the specific heat, AC,,, which has invariably been found to accompany the N + U transition (Sturtevant, 1977).It is, at least qualitatively,predicted from the exposure of hydrophobic residues in the process of unfolding, since the contact of such moieties with water is generally accompanied by large anomalous heat capacities (Tanford, 1973; Privalov, 1979; Privalov and Gill, 1988). In this context, detailed analysis of the unfolded states under different denaturing conditions is irrelevant because it is only the displacement from the native state that matters in analyzing protein stability. In contrast to previous claims, heat denaturation and complete unfolding (e.g., in 6 M guanidinium chloride) show the same characteristics as far as changes in heat capacity and enthalpy of unfolding are concerned (Tanford, 1968; Privalov, 1979, 1982, 1992). Thus, differences in the extent of unfolding cannot be significant. It is not clear to what extent an

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unfolded polypeptide chain actually conforms to the model of a random coil. Because there is no "good solvent" for both the backbone of a given protein and the chemically diverse side chains of its constituent amino acids, some nonrandom behavior is to be expected, not to mention the space requirements of the real chain (Tanford, 1968, 1970; Floly, 1969;Rose et al., 1985;Jaenicke, 1991b;Damaschun et al., 1991a,b). The molar enthalpy of protein denaturation, AH, at low temperature may either be positive or negative. Owing to the large ACp, it increases markedly with temperature. Considering the specific enthalpy, Ah+,u, instead of the molar enthalpy, the change in heat for compact globular proteins is found to converge at some temperature around 110°C. In cases where A H or Ah is positive, the conformational entropy, A P n f , must be the driving force in the overall denaturation reaction. Basically, A&-." is expected to be positive and to increase as far as alterations of the chain conformation on unfolding are concerned; on the other hand, the solvation term, Ashydr,which is attributable to exposure of hydrophobic residues and disruption of interior ion pairs, will be negative due to the ordering effects of charged groups and hydrophobic residues on water molecules in their surroundings. As in the case of the enthalpy of denaturation, the conformational entropy increases with temperature. Again, there is a tendency of A S + + ufor a great variety of proteins to converge at around 110°C. One may assume that at this temperature the increase in conformational freedom becomes the predominant factor, while the contribution of water to the entropy of denaturation vanishes (Privalov, 1989, 1992). On the basis of what has been mentioned for the temperature dependence of A H and AS, the free energy of thermal denaturation follows in a straightfoward manner: A(&.+" is a direct measure of the stability of the native macroscopic state under the condition that the free energy is calculated for an entire cooperative system. As mentioned in connection with domain proteins, this cannot be generally assumed, especially if complex proteins such as oligomeric or fibrous proteins are considered (Privalov, 1979, 1982;Jaenicke, 1991b). As illustrated in Fig. lA, at the temperature of maximum stability the entropy of denaturation vanishes so that the stabilization of N is determined only by the enthalpy term. At lower temperatures, A&-," becomes negative. As a consequence, TAS gains importance as the dominant structure-stabilizing factor. AHN+" also changes sign, but at still lower temperatures, this time promoting destabilization. Thus, the stability of the native state of a protein is achieved as the result of small shifts of the enthalpy and entropy functions along the temperature scale.

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A

B

Temperature ("C)

Temperature ("C)

FIG. 1 . Thermal stability of globular proteins. (A) Enthalpy, entropy, and Gibbs free energy of thermal unfolding of myoglobin. (B) Gibbs free energy of stabilization (ACN+L,), at pH values corrcsponding to optimal stability, for a-chymotrypsin (C), cytochrome c (Cc), lysozymr (1 .), metmyoglobin (Mb), ribonuclease ( RNasr), trypsin (T), and the (dimeric) basic pancreatic trypsin inhibitor (TI). AH, AS and A(; are given in kcal/mol, kcal/k . mol and kcal/mol, respectively (cf. Privalov, 1979).

Surprisingly, the superposition of the temperature dependence of AHN-ruand A&--." in the Gibbs-Helmholtz equation yields maximum curves (Fig. lB), suggesting that there must be a low-temperature analog of heat denaturation. A closer look at the data immediately shows that the low-temperature profiles extrapolate to a temperature range far below the freezing point, so that the N + U transition can only be reached in undercooled solutions or in the presence of denaturants (Nojima et al., 1977; Franks, 1985, 1995; Privalov and Gill, 1988). In fact, cold denaturation of proteins has been confirmed by a variety of experiments (Brandts, 1964; Pace and Tanford, 1968; Brandts et al., 1970; Damaschun t t al., 1993; Franks, 1995). The expectation that in the case of thermally stable proteins cold denaturation might become easily accessible at temperatures above 0°C has been disproved experimentally using proteins such as lactate dehydrogenase and glyceraldehyde-%phosphatedehydrogenase from T. mantimu (Jaenicke, 1981; Wrba et al., 1990a; Rehaber and Jaenicke, 1992). Considering the parabolic temperature profiles of AC,,,,, it is evident that enhanced thermal stability in thermophiles may be accomplished by three different mechanisms: as depicted in Fig. 2A, the profile of the mesophilic wild type (a) could either be lowered (b) or shifted to higher temperature (c), or it could be flattened (d). In all three cases, the "melting temperature" of the protein is shifted from the mesophilic denaturation transition T, to a thermophilic higher value T z . The few data that have been reported so far indicate that mechanisms (c) and (d) seem to prevail (Fig. 2B; Nojima el al., 1977, 1978, 1979; T. Oshima, personal communication 1994). In general, the temperature depen-

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A

189

B

40

0 LO 80 Temperature PCI

FIG.2. Temperature dependence of the Cibbs free energy of stabilization. (A) Hypothetical AGlrbprofiles for mesophilic (a) and thermophilic (b-d) proteins. T,* illustrates the shift of denaturation temperature in going from lower to higher thermostability. (B) Measured AC,,b for phosphoglycerate kinase from yeast (0) and Thennus thenophz1u.s (o), in the presence of 0.5 M (0) and 2 M guanidinium chloride ( 0 ) (data from Nojima et al., 1978).

dence of AGstabof thermophilic proteins seems to be less pronounced than that of the corresponding wild-type protein. Considering the maximum values in Fig. lB, it is striking that the free energy of stabilization, A(&+L,, of globular proteins with totally unrelated structures clusters in a narrow range between 30 and 65 kJ/mol (8-17 kcal/mol), independent of both the size of the proteins and the mode of denaturation (Pfeil, 1986). This small number corresponds to the equivalence of only few weak interactions, in spite of the fact that numerous noncovalent bonds are involved in the formation of the native secondary structure and the packing of the hydrophobic inner core. Their contributions compete with the decrease in configurational entropy and repulsive forces resulting from space-filling properties and charges, such that in summa the free energy of stabilization represents a small difference between large numbers. Calculating the stability increment per amino acid residue, AC,,, turns out to be one order of magnitude below the thermal energy. Thus, accumulating these increments in the process of structure formation would not overcome the thermal energy, in accordance with the observation that the overall stability of a polypeptide chain involves cooperativity. In cases such as bovine pancreatic trypsin inhibitor (BPTI ) or the designed four-helix bundle “felix” where the molecular mass of a protein is too small to provide the necessary size of a cooperative unit, covalent cross-links may contribute to stability by decreasing the entropy of unfolding (Creighton, 1978; Richardson and Richardson, 1989).

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In summary, the free energy of stabilization of globular proteins in aqueous solution is minute compared with the large numbers of attractive and repulsive forces involved in the formation of the densely packed native threedimensional structure. Stabilizing interactions compete with the decrease in configurational entropy. The biological significance of the low AG,,, is the requirement for balance between rigidity as a prerequisite of specificity, on one hand, and flexibility in connection with binding, activation, and release of substrates, etc., on the other. As has been mentioned, proteins may require covalent bonds such as disulfide bridges as additional stabilizing elements in order to maintain their functional state (Wetzel et al., 1990). There is no indication that cross-linking occurs as a natural stress response. In both mesophiles and thermophiles, cytoplasmic proteins commonly lack disulfide bonds. In principle, their building blocks are exclusively the canonical 20 “natural” amino acids. The unaltered repertoire of amino acids is in contrast to the range of variation of membrane lipids of (acido-)thermophiles and hyperthermophiles, which have been shown to contain a whole spectrum of unusual components (Langworthy and Pond, 1986). The distribution of amino acids in thermophilic proteins does not exhibit shifts that could be unambiguously correlated with specific a d a p tive responses to the various stress parameters. In the given context, differences in thermal stability cannot be correlated with differences in the cysteine/cystine content. As shown in Section IV, proteins from (hyper-)thermophiles do not exhibit properties qualitatively different from those of mesophilic ones. The essential adaptive alterations tend to preserve corresponding states under the mutual physiological conditions.

B. Fwces Involved in Proi!& Stabilization The threedimensional structure of proteins is determined by two classes of noncovalent interactions: electrostatic and hydrophobic. Electrostatic interactions include ion pairs, hydrogen bonds, weakly polar interactions, and van der Waals forces. Hydrophobic interactions imply van der Waals forces and hydration effects of nonpolar groups. Because of the superposition of significant enthalpic and entropic increments, their temperature dependence is complex and their contribution to protein stability the subject of some controversy (Dill, 1990; Pace, 1990; Privalov, 1992; Franks, 1995). Calorimetric measurements of the solution properties of nonpolar substances in water have led to the conclusion that the enthalpy of dissolution at room temperature is negative, with its absolute value pro-

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portional to the accessible surface area of the solute molecule. The corresponding heat capacity change is positive and, again, proportional to the solute surface area: it decreases with increasing temperature. The solution entropy is negative at 25°C and its absolute value decreases with increasing temperature. There are distinct differences between these transfer experiments and the thermodynamics of protein denaturation. In both cases, the enthalpy and entropy functions increase with temperature up to a limiting value above 1 10°C where “hydrophobic hydration” vanishes. The important difference refers to the transfer entropy, which is zero at this temperature, whereas the chain entropy is large and positive. This difference confirms earlier crystallographic and densitometric results which clearly indicated that the protein interior resembles a crystallike solid phase rather than a nonpolar liquid (Brandts, 1969; Hvidt, 1983;Kundrot and Richards, 1987). In the past, hydrophobic interactions have been commonly interpreted in terms of “entropic bonds,” that is, by the increase in entropy caused by water release (Kauzmann, 1959).However, high-precision calorimetry has clearly shown that there is a significant enthalpic contribution which may be ascribed to van der Waals forces (Privalov and Gill, 1988, 1989). At temperatures beyond about 120”C,AC,, converges and water becomes an “ordinary solvent.” It might not be fortuitous that both the upper limit of life and the temperature where hydrothermal degradation of amino acids becomes significant coincide with this temperature range (see Section 111). As has been mentioned, A&,, represents a marginal difference of large numbers as a consequence of the balance of attractive and repulsive forces. Attempts to calculate the threedimensional structure of proteins a6 initio by energy minimization have been unsuccessful just because of this compensatory effect. There is no way to develop confident and sufficiently accurate potential functions a6 initio. Thus, computer-aided predictions on the basis of known threedimensional structures are the only way to approach the problem. In this connection, limitations of the database have been the main reason for the limited success in the past (Rooman and Wodak, 1991,1992;Rooman et al., 1992);additional problems may arise from the fact that the empirical basis of the known threedimensional structures may be biased because it is restricted to crystallizable proteins. Approximate quantitative data with regard to the different types of intermolecular interactions have been obtained from known threedimensional structures. Keeping in mind that the increase in the free energy of stabilization, AAG,,, for extremophilic proteins is of the

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same order of magnitude as the overall free energy of stabilization, A&+,,, it is evident that minute structural alterations within a given protein molecule may suffice to cope with the various extremes of physical conditions. As a consequence, no general strategy in terms of preferred amino acid exchanges or specific types of intermolecular interactions is to be expected in going, for example, from rnesophiles to thermophiles (see Section V). Considering available structural and thermodynamic data, a number of conclusions may be drawn regarding the weight of the various types of interactions (Baldwin and Eisenberg, 1987; Dill, 1990; Pace et al., 1991;Jaenicke, 1991a,c). (i) A&+,, is equivalent to the energy required to break a maximum of five hydrogen bonds, corresponding to about 1% of the total number of hydrogen bonds in the folded structure of an average singledomain protein. (ii) In the unfolded state, a 10-kDa protein exposes about 440 polar sites, half of which are involved in internal hydrogen bonds in the native state. As a consequence, even a marginal difference in hydrogen bond strength between water-water and water-protein hydrogen bonds will be magnified to an energy change that may well exceed A(&+" (Pace et al., 1991). (iii) Water release from polar and nonpolar sites will lead to an increase in entropy, which is known to be the driving force in endothermic folding and assembly processes (Lauffer, 1975;Jaenicke, 1981, 1990). (iv) cy Hclices and extended P structures contribute significantly to protein stability (Jaenicke, 1991a); in this context, interaction of helix dipoles with charged groups in their vicinity may be important (Nicholson et al., 1991; Matthews, 1993). (v) Significant contributions may also be ascribed to multiple hydrogen bonds of the guanidinium group of arginine to backbone carbonyl oxygens (Borders et al., 1994). (vi) Only about 70% of the theoretically available hydrophobic contributions are realized as a consequence of the balance of favorable and unfavorable contributions to A&+,, on protein folding (Finney, 1982). (vii) As charged groups are commonly exposed to the aqueous solvent, intramolecular coulomb interactions cannot be of major importance in protein stabilization (Kauzmann, 1959; Dill, 1990): on average, only one ion pair per 150 amino acid residues in globular proteins is buried within the interior core (Barlow and Thornton, 1983). Thus, only surface ion pairs are expected to be involved in stabilization, in agreement with experiments using X-ray analysis and sitedirected mutagenesis (Perutz and Raidt, 1975; Hollecker and Creighton, 1983; Pace and Grimsley, 1988; Datr pin, et al., 1991a,c,d). Even for these groups, proton and water release may be more important than charge interactions (Stigter and Dill, 1990). (viii) Most polar sites in the inner core ofproteins are internally hydrogen

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bonded. Lack of this kind of internal saturation is found to be strongly destabilizing (Dao-pin, 1990). (ix) To bridge gaps between polar groups that are geometrically incapable of forming hydrogen bonds, in numerous cases otherwise separated polar groups have been found to be hydrogen bonded through so-called structural water molecules (Kuntz and Kauzmann, 1974; Finney, 1977). (x) In certain cases, ordered clathrate hydrates have been observed in the neighborhood of nonpolar residues exposed to the aqueous medium. Because a considerable amount of the accessible surface of proteins is hydrophobic (30-50%), extended areas of structured water may be found in the periphery of proteins (Lee and Richards, 1971; Teeter, 1990). Regarding the influence of temperature on the weak intermolecular interactions, the effects are highly complex. There is evidence from temperature dependence studies on systems other than proteins (e.g., surfactants and synthetic polymers) that for any system which can undergo order-disorder transitions, the temperature profile of the free energy of stabilization takes the form of a skewed parabola with the two characteristic transition temperatures illustrated in Fig. 2A (Franks, 1985). As mentioned, the phenomenon may be explained within the framework of the hydrophobic effect. ACp must change its sign at some limiting low temperature. In the case of proteins, the contributions to ACp come from both hydrophobic and coulombic interactions. Both become weaker with decreasing temperature, because of their entropic origin, on one hand, and the temperature dependence of the dielectric constant, on the other. Apart from this classic explanation, advances in the measurement and theory of hydration have shown that temperatureinduced recognition of polar rather than hydrophobic residues may be involved in macromolecular assembly owing to complementary patterns of surface polar groups. As shown by direct measurement of temperaturedependent intermolecular forces, the energies involved can be high (=kT per polar group, i.e., 0.6 kcal/mol; Leikin and Parsegian, 1994). Hydrogen bonds are favored at low temperature and become weaker as temperature is increased. Because of the compensatory effects in the total balance, predictions with respect to the significance of the various types of interactions cannot be made.

C. Structural Increments of th Free Energy of Stabilization Protein stability is accomplished by the cumulative effect of noncovalent interactions at many locations within a given molecule. In considering the contributions of local structural elements, protein fragments may be used in order to determine the minimum length of a polypeptide

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chain that is required to still form an intrinsically stable nativelike structure. Analysis by NMR has shown that oligopeptides down to six residues do form stable (nonrandom) conformations, supporting the idea that local structures may serve as “seeds” in the folding process (Wright et al., 1988). However, there is evidence that the short fragments do not necessarily adopt the same conformation in unrelated protein structures; for example, reverse turn motifs observed in small peptides seem to be absent in the known three-dimensional structures of proteins containing these sequences (Creighton, 1988).With regard to the stability of protein fragments, it has long been known that proteins are cooperative structures showing mutual stabilization of structural elements. Thermolysin has been used as a model to determine the fragment size at which nativelike structure is no longer formed. The N-terminal portion of the enzyme is found to stabilize the all-helical Cterminal domain which may be truncated to the 62-residue three-helix bundle without loosing much of its native (secondary) structure. In shortening the polypeptide chain, the free energy of stabilization drops steadily to half its value, while the temperature limit of denaturation is shifted from 87 to 64°C only. At a size of 20 residues, intermolecular interactions take over, leading to aggregation rather than proper structure formation (Vita d nl., 1989). The incremental stabilization observed for fragments holds also at the higher levels ofthe hierarchy ofprotein structure. Actually, the high intrinsic stability of fragments was the key observation which led to the concept thatproteinsconsistof “globules” (Goldberg, 1969)or “domains” (Janin and Wodak, 1983;Rossrnann and Argos, 1981) and undergo “folding by parts” (Wetlaufer, 1973; 1981;Jaenicke and Buchner, 1993).I n extreme cases, for example, in thermophiles, not only does the anomalous stability hold for the complete protein, but it can also be observed at the domain level (Jecht et nZ., 1994).A striking example illustrating the mutual stabilization of domains is the eye lens protein ykrystallin which consists of twoclosely similar 10-kDadomains,exclusivelyP sheets. The protein shows the bimodal denaturation transition, frequently observed for domain proteins, with the second phase coinciding with the unfolding of the isolated N-terminal domain (Rudolph et aZ., 1990).As for most eye lens proteins, the complete molecule is known to undergo n o degradation during the entire lifetime of an organism. The N-terminal domain shows this extreme intrinsic stability,whereas the Cterminal halfis surprisingly unstable, thus proving that domain interactions contribute significantly t o the overall stability of the protein. The mutual stabilization of domains in multidornain proteins holds also for the quaternary interactions of subunits in oligorneric or multimeric proteins. Their stabilizing effect has been clearly demonstrated

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in the case of the tetrameric enzyme lactate dehydrogenase, where the stability decreases steadily from the native 144kDa tetramer down to 1 4 and 21-kDa domain fragments. The tetramer, as a dimer of dimers, is highly stable, with a c1,, value of 2 Mguanidinium chloride (GdmC1) as the limiting concentration where deactivation occurs. The “proteolytic dimer” (with the N-terminal decapeptide cleaved o m requires structuremaking salts to exhibit activity; cl/2is found to be shifted to 5 1 MGdmC1. The monomer is inactive under any condition and is “structured” only as a short-lived folding intermediate on the pathway of reconstitution. The nicked NAD- and substrate-binding domains are unstable but still sufficiently well defined in their conformation to recognize one another and to pair correctly: in joint reconstitution experiments they lead to partial renaturation, exhibiting a mutual “chaperone effect” (Opitz et al., 1987). There are examples where stabilization due to subunit association goes to the extreme (viruses, chromatin, ferritin, bacterial surface layers, etc.). In all these cases, the monomers have average size, stability, and flexibility and perform their morphopoietic or catalytic functions, without being inhibited with respect to translocation, targeting, processing, etc. It is the assembly that is responsible for the anomalous stability and, consequently, the low turnover. The increments of protein stability discussed so far originate from the intrinsic properties of a given amino acid sequence. However, extrinsic factors not encoded in the sequence may also be of importance. For example, ions, cofactors, metabolites, o r components covalently linked to the polypeptide chain may affect both protein stability and folding. T o give an example, high levels of specific ions such as K+, cyclic 2,3diphosphoglycerate, or dimyoinositol 1,l ’-phosphate may enable nonthermostable proteins to gain enhanced stability (Hensel and Kijnig, 1988; Huber et al., 1989; Scholz et al., 1992). Similarly, polyamines such as caldopentamine and long-chain homologs were found in higher concentrations and with greater chain lengths as the growth temperature of T. mnn’tima was increased. At the maximum growth temperature, the tetraamine norspermine was reported to be the dominating protectant (Oshima, 1983; Zellner and Kneifel, 1993). The effect of protein conjugation has been investigated in detail, using yeast mutants with invertases differing in glycosylation. Here, the unglycosylated “internal,” the 34% “core-glycosylated,” and the 65% highly glycosylated “external” enzyme exhibit enhanced stability with increasing carbohydrate content (Kern et al., 1992a). As shown by the fact that the glycosylated forms of the enzyme do not undergo heat aggregation, the glycomoiety, apart from stabilizing the protein, has a

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significant solubilizingeffect. This fits the observation that the chaperone action of GroEL is observed only for carbohydrate-free, internal invertase, whereas the glycosylated forms of the enzyme do not interact with the chaperone (Kern et al., 1992a,b).

D. What Model Studies Teach about Thermal Stability

As the structural basis of protein stability is still unresolved, no clearcut correlations are available to understand the thermal properties of a given protein. Therefore, in this section, some representative examples are discussed that may serve as models to illustrate how stability is brought about in cases where well-defined local changes in structure allow a detailed analysis. In trying to relate stability to specific structural elements in proteins, maximum stability has been observed in all*, all-& and crp proteins (e.g., rop, &crystallins, immunoglobulins, and oligomers such as NAD-dependent dehydrogenases) .Sitedirected mutagenesis has been the method of choice in determining the contributions of specific groups or intermolecular interactions to the structure and intrinsic stability of a model protein. 1. Bac2eriOphage T4 Lysoryme

Bacteriophage T4 lysozyme consists of two functional domains that show pronounced interdomain rotation, allowing the active-site cleft to bind the oligosaccharide substrate (Weaver and Matthews, 1987). The overall conformation of the molecule is mainly a helical with short stretches of psheet strands in the N-terminal domain. Wild-type and mutant T4 lysozyme have been used to study the effect of local exchanges at structurally relevant sites of the enzyme (Matthews, 1987, 1991, 1993; Alber and Matthews, 1987; Alber et al., 1987a,b, 1988; Alber, 1989a,b; Karpusas et al., 1989; Dao-pin, 1990; Dao-pin et al., 1990,1991a-d; Zhang el al., 1991). To give a few examples, a hydrogen bond at Thr-157 is found to contribute significantly to stability, whereas mutations at Pro86, in spite of an increase in helicity, do not have a significant effect. Obviously, local improvements are compensated by remote distortions of the structure. Similar experiments referring to the effect of hydrophobic interactions concentrated on Ile-3, where alterations in stabilization were found to be proportional to the surface area of the altered hydrophobic residues. In this connection, improved hydrophobic packing by filling cavities caused marginal destabilization, again showing that the gain in local stabilization can be offset by the introduction of strain (e.g.. by nonoptimal dihedral angles). In cases where exchanges disrupt the contact

STRUCTURE AND STABILITY OF

HYPERSTABLE PROTEINS

197

region between helices, extreme destabilization is observed. On the other hand, reduction of strain within an a helix increases stability. In going one step further, the helix stabilizing properties of Ala have been used to “simplify” the enzyme by replacing non-Ala residues in an a helix; the stepwise substitution yields a significant increase in AAGslab which is to a first approximation additive. Interactions of helix dipoles with charged groups are highly significant; for example, introducing an acidic group close to the amino terminus of an a helix is one of the most consistently effective ways to increase protein stability (Nicholson et al., 1991; Matthews, 1993). As mentioned in connection with the stabilizing effect of external salt linkages in thermophilic ferredoxin and certain hemoglobins (Perutz and Raidt, 1975),engineered ion pair interactions may have a stabilizing effect depending on distance, pH, and ionic strength. At neutral pH and a distance around 3 A, a slight stabilization is accomplished; beyond 3.5A the effect vanishes. Finally, as one would predict, burying “charged residues” in the hydrophobic core region has a dramatic destabilizing effect (see Jaenicke, 1991a). The active site of T4 lysozyme lies in the loosely packed cleft between the compact domains, suggesting that “functional” amino acid residues are optimized for flexibility rather than stability. Actually, mutating residues involved in either catalysis or substrate binding is found to enhance stability at the cost of reduced activity, supporting a relationship between stability and function (Fig. 3; Shoichet et al., 1995). In all cases that have been briefly described, the enzyme was shown to be isomorphous, that is, the structure was only locally perturbed. N o example has been worked out in as much detail. The mosaic that has emerged illustrates how subtly the different weak interactions are balanced in native globular proteins, and how much even marginal local strain in the threedimensional structure may affect protein stability. From the point of view of protein engineering and the attempt to provide deeper insight in to the mechanisms of thermophilic adaptation, data accumulated for lysozyme T4 as a test case stress the conclusion that at present unambiguous predictions or general strategies of protein stabilization cannot be given. 2. Oligomers With the conclusions from the lysozyme data in mind, explanations of the stability of oligomeric or multimeric proteins can at best be hypothetical. That subunit association (as domain interactions) may drastically improve protein stability has been mentioned (Section I1,B). In the case of lactate dehydrogenase, Rossmann and co-workers were

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h

8 3 .> .c

100

-

.-9

-

50

-

3

0

v

0

m

44

+

Catalysis

(D

2

Y

-+-+-+-+-+-+-+-+-+-=-+

S-Binding

iJiii

+

+-+-

---hr.r.sC

vlv)vIv)

FIG.3. Comparison of the stability (top) and activity (bottom) of wild-type (W)and mutant T4 lysozymes with substitutions in residues involved in catalysis (Gly-I 1 and Clu20) and in substrate binding (Sbinding, Ser-117 and Asn-132). Stabilitirs and relative activities are calculated relative to the native wild-type protein, with positive values indirating variants more stable than wild type. Asterisks (*) mark active-site mutants. Data from Shoichet el al. (1995).

able to give a detailed account of the weak intermolecular interactions involved in the assembly (Holbrook d al., 1975). As one would expect, the decrease in hydrophobic surface area is of major importarice; however, other types of bonds do participate. For example, the sensitivity of subunit dissociation with respect to pH and ionic strength proves charge interactions to be involved. “Grafting” of mesophilic lactate dehydrogenases with domains of thermophilic homologs to form chimeras with substructures of varying thermal stability confirms independent folding and suggests conservation of the intrinsic stabilities of local structures (Waldvogel et al., 1987; Znlli et al., 1991; Biro et al., 1990; Toma et al., 1991). Suggestive as such experiments might be, they do not allow unequivocal conclusions with respect to the correlation of

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structure and stability as long as the three-dimensional structures of the constructs are unknown. Sequence alignment and homology modeling can still not replace experimental structure determinations (Fasman, 1989). Considering point mutations that result in proteins with increased thermal stability (known to be isomorphous and practically unaltered in their enzymatic properties), effects closely similar to those discussed in connection with the stability of mutants or lysozyme T4 are observed. For example, single, double, and triple mutants of creatinase (a homodimer of 110 kDa molecular mass) exhibit additive increments of stabilization, caused by a decrease in hydrophobic surface area and improved packing density; changes in subunit interactions do not play a significant role (Schumann et al., 1993).In contrast, in the case of pyruvate oxidase, thermostable point mutations cause improved coenzyme binding and subunit association, thus promoting the native tetrameric quaternary structure (Risse et al., 1992a,b). E.

Kinetics

The space-filling properties of the amino acid residues involved either in the packing of the native polypeptide chain or in the solvent-accessible surface area of globular proteins have important kinetic implications. Considering the planarity of the peptide bond, only the torsion angles around the N-C, and the C,-C bonds (4 and +) are at disposal. With the given van der Waals radii of the amino acid side chains, this reduces the allowed regions in the Ramachandran +,$diagram of free rotations and energy minimization to less than 7% (Dickerson and &is, 1969; Br2ndCn and Tooze, 1991). As a consequence, the time required to search for the native conformation as the “kinetically accessible state of minimum free energy” is reduced significantly, shifting Levinthal’s paradoxical time requirement for the structure formation of proteins from an astronomical to a biologically feasible time range (Baldwin and Eisenberg, 1987). Further enhancement comes from the fact that protein folding proceeds along a well-defined pathway (instead of being a random search process); side reactions do contribute to the overall reaction, but at much lower yield. The elementary processes involved are exceedingly fast: both a-helix and @structure formation show half-lives in the microsecond time range (Schwarz and Engel, 1972; Briggs and Roder, 1991). In all+ structures, folding seems to be slowed drastically because of the importance of long-range interactions between the strands (Varley et al., 1993).

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In dealing with kinetic aspects of temperature adaptation, one needs to consider Arrhenius effects on metabolic reaction sequences, apart from stability and folding. It is obvious that altered temperature conditions in a given habitat require both directed alterations of the intrinsic stability of all cell constituents and tuning of the kinetics. In connection with metabolic pathways, a complete understanding of temperature adaptation can be achieved only if one’s field of vision encompasses all the interacting components, the cellular microenvironment, the enzymes, and the membrane lipids with which they interact. As Somero (1978) pointed out, this implies that unless we reproduce in vitro the cellular conditions, the scope and nature of molecular adaptation may not be discerned. Depending on the activation energies of the reactions involved, shifts in the optimum temperature from mesophiles to thermophiles may cause dramatic kinetic dislocations owing to differences in the activation energies involved in the metabolic network (Table I). In this context, two points are crucial: (i) temperature adaptation is synonymous with the achievement of “temperature compensation” of metabolism whereby the rates of metabolic processes are adjusted to offset the accelerating or decelerating effects of temperature changes; (ii) owing to the fact that correct binding abilities for ligands are essential for initiation and regulation of catalysis, ligand binding properties must be strongly conserved. The underlying experimental observations are conservation of I%, values (Somero, 1978) and enthalpy-entropy compensation observed for the temperature dependence of the free energy of ligand binding (Jaenicke, 1981). 111. LIMITSOF GROWTH

So far, protein stability has been discussed on the basis of free energy changes determined from N Ft U equilibrium transitions in various denaturants. The corresponding data were compiled assuming reversibilTABLE 1 Alterations of Relative Reaction Rarcs Normalized to 20°C Temperature

(“C) 20 60 100

Activation energy (kJ/mol) 16

32

64

1 3

1 10 186

1 90 30,000

14

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

20 1

ity, that is, absence of irreversible denaturation such as heat aggregation or covalent modifications of the polypeptide chain. That this tacit a s sumption does not hold under extreme conditions is obvious. Therefore, in this section a brief account of the chemical stability of proteins and their constituent natural amino acids has to be given.

A.

Limits of Growth uersus Limits of Thermal Stability: Model Studies Considering the limits of growth in the biosphere, it is known that proteins at temperatures beyond 100°C and pressures of the order of 100 MPa ( 1000 atmospheres) undergo covalent modification. In devising model experiments to get insight into the chemical mechanisms, one has to keep in mind that the building blocks of proteins from extremophiles are exclusively the canonical 20 natural amino acids. Thus, thermophiles must compensate for their degradation by enhanced synthesis, or they must have evolved protection or repair mechanisms. Apart from the oxidation of cysteine and methionine, and the deamidation of asparagine and glutamine, little is known about the detailed chemistry involved in the hydrothermal degradation of amino acids and peptides, even less about protection and repair. In the case of conjugated proteins such as glycoproteins, the Maillard reaction may serve as a paradigm to illustrate the complexity of the chemistry involved (Barrett, 1985). In studying the physical conditions at the borderline of life, for example, in volcanic areas in the deep sea, one may predict that most biomolecules will undergo hydrothermal decomposition. To define the upper temperature limit of protein stability, amino acid mixtures, polyglycine, and cells of the hyperthermophilic archaeon Pyrodictium occultum ( T,,, 110°C) were subjected to temperatures between 20 and 250°C and pressures up to 26 MPa (260 atmospheres). At 250°C and 26 MPa, Asp, Glu, Ser, Thr, Cys, arid Trp are fully decomposed, whereas apolar amino acids as well as His, Lys, Arg, and Phe undergo partial degradation. On the whole, the transformation leads to a drastic increase of Gly, Ma, and ammonia, in agreement with the end products of geochemical processes (Vallentyne, 1964) (Fig. 4A).The effects are closely similar, independent of whether water at pH 2.0 or phosphate buffer, pH 7.6, is applied as solvent. Judging from the temperature profiles (Fig. 4B), the increase in thermal stability follows the series Cys, Arg, Glu, Asp < Trp < Ser < Thr < Met < His < Lys < Tyr < Ile < Leu, Val. In all cases, the halflife of the reactions is significantly shorter than the generation time of most hyperthermophilic microorganisms. Using polyglycine as the most stable polyamino acid, complete hydrolysis of the peptide bond under the above extreme conditions is accomplished within a time range of

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A

B

8 3 %&%BE!aZ&

2-a

5 4

3 2 1

0

1

Temperature (“C) FIG 4. Hydrothermal degradation of natural L-amino acids at 250°C and 26 MPa. (A) Solid lines denote the standard mixture of 0.5 pmol of each amino acid without hydrothermal treatment; dotted lines denote the mixture in 30 mM HEPES/60 mM NaHCO,, pH 7.5; filled circles show the mixture in water, pH c4.7. Numbered peaks (1-5) are unidentified degradation products. (B)Temperature dependence of the decomposition of natural L-amiiw acids in 0.1 M sodium phosphate bufi‘er, pH 7.0, after 6 hr of incubation at 26 MPa. The ordinate gives peak areas in arbitrary units (Bernhardt el al., 1984).

minutes. In the case of alanylalanine and alanylaspartate, the half-life of hydrolysis is -6 min and < 1 min, respectively. Whole cells of Fyrodictium occulturn show the same characteristics described for amino acid mixtures, proving that even the most extreme thermophile presently known does not show any property suggesting the occurrence of specific constituents, either intrinsic or extrinsic. All the above-men tioned decomposition reactions are too fast to be compensated by enhanced resynthesis (White, 1984). In this context, one important limiting factor for the viability of microorganisms is the hydrolysis of ATP. An investigation of the stability of ATP in aqueous solution (Leibrock el al., 1995) clearly supports the view that the ultimate temperature limit of ATP-dependent metabolism lies between 110 and 140°C. Within this temperature range, heat-sensitive biomolecules could possibly still be resynthesized at biologically feasible rates (Stetter et al., 1990). The occurrence of abyssal hyperthermophiles that grow and multiply in the hostile deep-sea environment demonstrates that still unrecognized stabilizing mechanisms must exist. As enzymes taken from the bacterial genera AguifeJc and Thermotoga or the archaeal genera Acidinnus, Archaee globus, Desulfurococcus, Desulfurolobus, Methanococcus, Methanothamus, MPL-

STRUCTURE AND STABILITY OF I-M’ERSTABLE PROTEINS

203

allosphaera, &-ococcus, Fyrodictium, Staphylothermus, Stygioglobus, Sulfolobus, Thermococcus, Thennodiscus, etc. (Stetter, 1992,1993),show intrinsic stability up to the limits of their respective maximum growth temperatures, the strategies must have evolved mainly at the protein level. That stabilization by extrinsic factors and compatible solutes may shift the thermal denaturation of proteins and the maximum growth temperature of prokaryotes as well as eukaryotes to higher temperatures has been known for a long time (see Pfeil, 1986; Carpenter et al., 1993; for specific stabilizing compounds that occur in thermophiles, see Section I1,C). In discussing the temperature limit of the chemical stabilityof proteins, it is interesting to note that the onset of thermal degradation coincides with the temperature range where the hydrophobic hydration of proteins vanishes (Baldwin, 1986; Privalov, 1990, 1992). Because thermophilic proteins consist exclusively of the common 20 natural amino acids, we may conclude that it is their integrity, together with hydrophobic interactions, which determines the upper temperature limit of growth. The hypothesis that the ultimate requirement for life is the presence of liquid water, and that its occurrence may be considered the necessary and sufficient condition for biological function, cannot be correct. It holds at extremely low temperatures or in the dry state, that is, at water contents below the normal level of protein hydration (<0.25 g H 2 0 / g protein), where viability is preserved by “cryptobiotic” adaptation. However, attempts to expand the limit of viability at high temperatures by stabilizing the liquid state of water at increased hydrostatic pressure failed: There is no shift of the maximum growth temperature of the moderately thermophilic archaeon Methanococcus thennolithotrophicus if the pressure is increased up to 50 MPa (Bernhardt et al., 1988;Jaenicke et al., 1988); thus, so-called black smoker bacteria are science fiction rather than biological science (Bernhardt et al., 1984). From the chemical point of view, irreversible thermal denaturation includes a wide variety of reactions: cystine destruction by blimination, thiol-catalyzed disulfide interchange, oxidation of cysteine and methionine, and deamidation of asparagine and glutamine residues. Close to neutrality, these five processes appear to demarcate the upper temperature limit of protein stability. At pH 4.0 or below, in addition, hydrolysis of specific peptide bonds (especially at aspartic acid residues) gains importance (Zale and Klibanov, 1986; Tomazic and Klibanov, 1988; Volkin and Klibanov, 1992). It is obvious that cellular components and functional groups other than proteins and their constituents may participate in a whole spectrum of chemical processes, including Maillard and group transfer reactions, lanthionin formation, covalent crosslinking, etc.

204

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It is tempting to inquire whether nature has followed adaptive strategies by avoiding specific reactive residues or “weak links” in protein molecules in order to keep the native sequence intact. Comparison of amino acid compositions of mesophilic and thermophilic microorganisms revealed no such evidence (Singleton and Amelunxen, 1973;Argos et al., 1979;Jaenicke, 1981). Considering the growth characteristicsof thermophiles at low temperatures, certain bacterial and archaeal genera turn out to be so well adapted that they do not multiply at temperatures below 60 or even 80°C: (Stetter et ab, 1983). It is dimcult to give a clear-cut explanation for this observation since energetic and kinetic effects may be involved. In contrast to psychrophiles where thermal instability of proteins has been shown to coincide with the upper limit of viability (Adler and Knowles, 1995),cold denaturation cannot be of importance. The same holds for temperature effects on ligand binding and protein folding. Taking the formation of enzyme binary and ternary complexes as a first example, no significant temperature dependence of the free energy of ligand binding occurs due to enthalpy-entropy compensation (Fig. 5 ) (Hinz and Jaenicke, 1975; Schmid et al., 1976). With respect to protein folding, the production of recombinant enzymes from hyperthermophiles in Escherichia coli clearly shows that heterologous expression at ambient temperature yields functional protein; in the case of Tm glyceraldehyde-%phosphatedehy-

lo 20 30 LO TOC

l o 20 30

LO

--

FIG.5. Enthalpy-entropy compensation on ligand binding to pig muscle lartate dehydrogenase. (For experimental details, see Hinz and Jaenicke, 1975.)

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

205

drogenase (GAPDH), the temperature difference between the host and guest species is close to 60°C (Tomschy et al,, 1993). Keeping in mind that the forces involved in protein stabilization are critically temperature dependent, this observation is by no means trivial. Actually, on refolding at low temperature (
Before entering the more specialized discussion of the solution properties and self-organization of glycolytic enzymes from the hyperthermophilic bacterium T. maritima, it might be appropriate to give a short survey of possible mechanisms of thermal adaptation. Considering the potential maximum stability of polypeptides, studies on synthetic polypeptides have shown that the repertoire of the 20 natural amino acids basically allows the generation of protein molecules with stabilities beyond those commonly observed for natural proteins (de Grado, 1988). Obviously, evolution results in biomolecules with optimum function rather than maximum stability. In cases such as surface layers of acidothermophiles or the tubular network of brodictium, both requirements coincide, leading to absolute records of protein stability: the proteins preserve their structure even in strong denaturants (H. Kenig, personal communication 1996; Rieger et al., 1995). The molecular basis of the stability of these systems is still unknown. Since the early 1980s, mutations underlying the thermal adaptation of structural genes of proteins from thermophilic microorganisms have been assumed to be manifested in certain welldefined shifts in the amino acid composition. Because the comparisons were based on either functionally unrelated proteins o r a too small a sample size, the significance of the observed preferences was doubtful (Jaenicke, 1981). Comparing the sequences of ferredoxins, glyceraldehyde-%phosphate dehydrogenases, and lactate dehydrogenases from mesophiles and thermophiles and their corresponding threedimensional structures, Argos

206

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et al. (1979) proposed rules for the temperaturedependent “gross traffic” of amino acids in going from mesophilic to thermophilic sequences. They suggest that relevant contributions to thermal stabilization may be attributed to both enhanced helicity (e.g., Ser + Ala, Val -+ Ala) and hydrophobicity (Gly -+ Ala, Ser +Ala, Ser -+ Thr/Gly/Ala, Asp -+ Asn/ Glu), as well as the exchange Lys + Arg. Argos’ rules were later refined on the basis of data from a larger number of protein sequences and structures (MenCndez-Ariasand Argos, 1989). Decreased flexibility and increased hydrophobicity, both preferably in a-helical regions and to some extent in domain interfaces, were suggested as the main stabilizing principle. The ranking of the five most frequent amino acid exchanges from mesophiles to thermophiles was now seen to be Lys + Arg, Ser + Ala, Gly -+ Ma, Ser -+ Thr, and Ile -+ Val. Decreased sidechain flexibility was also proposed by Janin et al. (1978); in their analysis, improvements in the internal packing of residues are considered to be less important. As will be shown, the significance of most of these rules is ambiguous because the low extra free energy of thermal stabilization can be accumulated by innumerable combinations of subtle changes of local weak interactions. So far, generalizations in terms of preferred amino acid exchanges, or variations in secondary structure, etc., have been unsuccessful (Jaenicke, 1991a).Attempts to develop algorithms to discriminate between normal and anomalous structures of proteins (in terms of correlation functions with confidence limits) have provided methods to a p proach the problem in an objective standardized way (Bdhm and Jaenicke, 1992, 1994). In this context, the assessment of the accuracy of homology modeling at a given level of sequence identity is critical. In studying the limitations of the method, Hilbert el al. (1993) have shown that in comparing sequences with less than 50% identity, deviations in structurally not conserved regions continually increase. This implies that with decreasing sequence identity, modeling has to take into consideration more and more structurally diverging loop regions that are difficult to predict. In cases where functional requirements (cofactor o r substrate binding, etc.) cause significant structural effects, predictions become definitely ambiguous. Considerations of this kind are relevant in comparing the sequences of mesophilic and extremophilic proteins in order to extract rules of molecular adaptation. What seems to be established with respect to thermophiles are the following observations. ( 1) Proteins from thermophiles commonly exhibit high intrinsic stabilities that become marginal at their respective maximal growth temperatures. At the temperature of maximum activity, homologousenzymesshowasimilardegree ofconformational flexibility. (2) Avail-

STRUCTURE AND STARILITY OF HWERSTABLE PROTEINS

207

able structural data prove that thermophilic proteins are closely similar to their mesophilic counterparts regarding basic topologies and enzyme mechanisms. (3) The correlation of sequence alterations with changes in thermal stability is highly complex. The increments of stabilization o b served for single, double, and multiple point mutations are approximately additive, corroborating the idea that thermal stability is the cumulative effect of small improvements at various locations within the core of the protein or between its subunits (Jaenicke, 1991a; Risse et al., 1992a,b; Schumann et al., 1993). (4)In numerous cases where high-resolution structures of point mutants are available, n o clear-cut correlation between the increase in stability and alterations in the threedimensional structure can be found because the crystallographically determined differences between mutants [root mean square (rms) 0.2 A] are not focused at the exchanged residues but spread over larger portions of the molecule (R. Huber, personal communication 1996). ( 5 ) No general strategies of thermal adaptation can be given, except for the (rathervaguely defined) p r o p erties deduced from correlation functions: enhanced hydrophobicity, reduced surface area, and increased packing density of the interior core of the molecule (B8hm and Jaenicke, 1992).

IV. HWOTHERMOPHILIC BACTERIUM Thamotoga Mantima Members of the order Thermotogales thrive within active geothermal areas, from which they were originally isolated (Huber et al., 1986; Huber and Stetter, 1992; Stetter, 1993). They occur in marine hydrothermal systems as well as in low salinity solfataric springs. An isolate from an oil-producing well has also been reported (Ravot et al., 1995).From a phylogenetic point of view, the Thermotogales represent (next to AquifeJc)the deepest branch and the most slowly evolving lineage within the bacterial domain and include the most extreme thermophilic bacteria presently known (Woese, 1987). This property, together with the growing knowledge of the genome of the species T. maritima (Kim et al., 1993),and the fact that the organism can be cultivated in large-scale fermentations, made the bacterium one of the most actively investigated objects in thermophile research. Thamotoga muritima grows in the temperature range between 55 and 90°C with an optimum at around 80°C. The rod-shaped (-2 X 0.6 p m ) gram-negative bacterium is surrounded by a sheathlike outer envelope, the toga, that balloons over the ends of the cell body and consists of a porin-type trimeric protein (Rachel et al., 1990). Sensitivity to lysozyme indicates the presence of a murein cell wall. As in other bacteria (Vihinen and M5nts212, 1989), amylases seem to be tightly bound to the toga;

208

R. JAENICKE ETAL.

further complex formation has not been observed. The bacterium is a strictly anaerobic fermentative organotroph that grows on various sugars, cellulose, starch, and glycogen as the carbon source. Starting from simple sugars (glucose, ribose, xylose) and complex carbohydrates as the energy source, the metabolism predominantly yields L( +)-lactate, acetate, C 0 2 , and molecular hydrogen (Huber et aL, 1986) (Fig. 6). Because high levels of H, inhibit the growth, the gas must be removed by either flushing with N2 or argon or by adding elemental sulfur (So) which is reduced to H2S.Thus, So serves as an oxidizing agent in H2detoxification. Evidently T. muntimu is unable to gain additional energy from So respiration (Huber and Stetter, 1992).The organism also grows well on complex media containing tryptone and yeast extract. A peptide source is required for growth on carbohydrates since the organism does not utilize ammonia or free amino acids as the N source (Adams et al., 1992). Systematic screening for significant enzyme activities in crude extracts of T. mantima provided clear evidence that glucose fermentation mainly follows the conventional Embden-Meyerhof glycolytic pathway and, to a lesser extent, the Entner-Doudoroff pathway and the citric acid cycle (V. Krivenko, 1988). An accurate investigation of the stoichiomeuy of glucose fermentation revealed that 1 mol glucose is fermented into 2 mol acetate, 2 mol COP,and 4 mol H,; the yield of ATP was 4 mol/ mol of glucose (Schr6der et al., 1994). Cell extracts contain L-lactate dehydrogenase activity (Wrba el al., 1990b; Ostendorp et al., 1993) but at very low levels, so that under normal growth conditions lactate formation cannot serve as an electron sink reaction. Furthermore, enzymes that are involved in acetate formation from pyruvate [pyruvate :ferredoxin oxidoreductase (POR, pyruvate synthase), phosphate acyltransferase, and acetate kinase] and in H4 formation (NADH: ferredoxin oxidoreductase and hydrogenase) are present (Schrdder d al., 1994). Both POR and the hydrogenase from T. mantima have been purified and studied in detail (Juszczak et al., 1991; Blarney and Adams, 1994; Smith d al., 1994). The POR enzyme generates acetylCoA and C02 from pyruvate, with ferredoxin as electron acceptor for the ultimate H, production via hydrogenase. Surprisingly, the activities of both enzymes are stimulated by tungsten, which also enhances the growth of the organism. The enzymes do not contain the metal, and the physiological role of tungsten remains enigmatic. Ferredoxin has been isolated, cloned, and expressed in E. cob; the 6.2-kDa protein contains a single [4Fe-4S] iron-sulfur cluster which is correctly incorporated during heterologous expression in E. coli (Darimont and Sterner, 1994).The three-dimensional structure of the protein has been modeled

209

STRUCTURE AND STABILITY OF HWERSTABLE PROTEINS

STARCH Amyk

1 1

MALTOSE

Glueodduc

ATP

ADP

4-

GLUCOSE

4

Glucose 6-phosphate

FNCIOSC 6-phosphate ATP

ADP

7j

-/1

FNCIOSC I.&bisphosphate

A -

1F'L% ; 2ADpy Glyccraldchydc 3-phosphalc

Dihydroxyacctone phosphate

2 NAD*

4 H*

CAPDH

2 NADH 2 x 1.3- Bisphosphoglycerate

PCK

2 ATP

HYdrgQ=

2H2

2 x 3-Phosphoglycerale

1 1

*

2 a 2-Phosphoglyceras

ENOLASE

-1

Aminoacids

-Y

2 x Phosphoenolpyruvate

@ROTEINSIPEFTIDES

2 ATP 2ADP

-- - - - ---

2 x Pyruvatc

POR 2CoA

2 co*

LDH 2NADH

2NAD+

2

2 Fdox

2F d d

2 XLaClalC

14H Hydmgewc

2 H2

2 a Acelyl-CoA

I

ADP 2 j a y l p h o s p h a i c

2 ATP 2 a Acelalc

FIG.6. Metabolic pathways of carbohydrate fermentation in T h o t o g a muntima. After Schrdder ~t nl. (1994).

210

R. JAENICKE ETAI..

(Doelz et al., 1994) and subsequently confirmed by NMR (P. R6sch, personal communication 1996). For the degradation of proteins and oligomeric carbohydrates, T. maritima contains a number of proteases and saccharolytic enzymes. Surprisingly, neither amylases nor proteases are detectable in the growth medium (Schumann et al., 1991; H. Schurig, unpublished results 1992). As in other bacteria, amylases seem to be bound to the cell surface. Percoll gradient centrifugation shows that more than 85% of the total a-,p, and glucoamylase activity are associated with the toga. Evidently, instead of secreting the enzymes and taking up glucose, the organism degrades starch on the cell surface. The level of expression of the three amylase isoenzymes is too low to isolate the proteins in quantities required for a detailed physicochemical characterization. Compared with a-amylase from Bacillus lichenifonnis ( T , 75"C), the Tm amylases show high intrinsic stability, with upper temperature limits for catalysis beyond 95°C. As might be expected, significant turnover of the Bacillus and Thermotoga enzymes requires physiological temperatures. For example, their activity ratios Amax/Ae50care -2 and >loo, respectively. The molecular masses and the enzyrnological properties of the homologous enzymes from other organisms are similar (Schumann el al., 1991). Thennotoga maritima also contains an enzyme, 4.cu-glucanotransferase (EC 2.4.1.25). involved in the breakdown of 1,h-glucans (i.e., starch, amylose, and amylopectin) . This monomeric 53kDa enzyme disproportionates 1,4maltodextrins by glycosyl transfer of polymers to saccharides smaller than about 12 glucose residues. Its gene has been cloned and expressed in E. coli. With a transition temperature of 85°C the native 7'. maritimn enzyme represents the most thermostable glycosyltransferase described so far (Liebl et al., 1992; Heinrich et al., 1994). The occurrence of P-galactosidase (EC 3.2.1.2 1) allows T. maritima to grow on low molecular weight degradation products of cellulose (cellobiose) as a carbon source. The quaternary structure of the pure enzyme varies from dimers to tetramers and octamers, with a subunit molecular mass of 47 kDa. It exhibits high intrinsic thermal stability (up to 95°C) and maximum activity at 90°C (pH 6) (Gabelsberger et al., 1993; Liebl et al., 1994). The metabolic breakdown of xylan, the main component of hemicellulose, requires the action of endo- 1,4-@xylanase (EC 3.2.1.8). In this case T. muritimu (strain FjSSSB.1) makes use of xylanase secretion in order to utilize the substrate. The enzyme, with a pH optimum at pH 5.4, shows thermal characteristics similar to those reported for amylase. Over a 20-min assay, maximum activity is observed at 105°C; immobilization and addition of sorbitol allow the temperature range to be extended to about 120°C (Simpson et al., 1991). The gene

21 1

STRUCTURE AND STABILITY OF HWERSTABLE PROTEINS

for the enzyme from T. maritima strain MSBS was cloned in E. coli and expressed as an intrinsically thermostable, soluble enzyme. Sequence analysis suggests that the 120-kDa polypeptide consists of five domains, of which the central one contains the catalytic site (Winterhalter et al., 1995). When grown with xylose as the sole carbon source, T. man'tima is able to induce &glucose isomerase (EC 5.3.1.5, xylose isomerase), a homotetramer with 45-kDa subunits and a typical requirement for metal ions (Mg'+, C o y + ,or Mn2+).The ions are involved in both substrate binding and quaternary structure maintenance. At the pH optimum (pH 7.5), the enzyme has an optimum temperature for catalysis of 105°C. The catalytic properties clearly show the preference of the enzyme for xylose over other sugars (Brown et aL, 1993). V. GL.YCOLWIC ENZYMES As mentioned, T. man'tima gains metabolic energy by substrate-level phosphorylation rather than by respiration (Table 11). The activities in TARLE 11 Specific Activities and Apparent K, Values of Enzymes Involved in Glucose Metabolism" Enzyme activity Hexokinase

0.29

GlucoseCFphosphateisomerase Phosphofructokinase

0.56 0.19

Fructose-I ,Bbisphosphate aldolase Triose-phosphate isomerase Glyceraldehyde-%phosphatedehydrogenase Phosphoglycerate kinase

0.03 6.30 0.22 (NADt) 0.004 (NADPt) 3.70

Phosphoglycerate mutase Enolase

0.40 4.00

Pyruvate kinase

0.05

Phosphate acetyltransferase Acetate kinase Hydrogenase

0.15 (80°C) 0.2 15.00 (80°C)

1.1 (Glc) 1.6 (ATP) 0.5 (C-6-P) 2.8 (F-6P) 0.09 (ATP) 0.02 (FBP)

-

3.9 (P,) 0.06 (NAD') 1.0 (3PG) 0.31 (ATP) 3.3 (SPG) 0.18 (2-PG) 0.26 (MgSO,) 0.06 (PEP) 0.18 (ADP)

In cell extracts of 7'. maritima (50°C). Data from Shrdder et al. (1994).

-

-

212

R JAENICKE ETAL.

cell extracts of various glycolytic enzymes vary over a wide range. For example, the expression level of glyceraldehyde-%phosphatedehydrogenaSe (GAPDH) is more than 1000-foldcompared with the level of lactate dehydrogenase (LDH). Triose-phosphate isomerase (TIM) activity is detectable only in combination with phosphoglycerate kinase (PGK); as discussed below, the two enzymes form a covalent complex. Presently, five enzymes of the glycolytic pathway have been purified and at least partially characterized. These are GAPDH, TIM, PGK, enolase, and LDH (Wrba et al., 1990a,b; Rehaber and Jaenicke, 1992; Ostendorp et al., 1993;Jaenicke, 1994a). All five enzymes show anomalously high intrinsic stabilities, with denaturation temperatures above the optimum growth temperature of the bacterium. For example, GAPDH undergoes an irreversible thermal transition at 109OC, which is the highest observed so far for a bacterial protein (Wrba et al., 1990a; Huber and Stetter, 1992). The glycolytic enzymes are highly homologous to their mesophilic counterparts, in terms of sequence, topology, and enzymatic properties. On the other hand, each of the enzymes exhibits some unexpected peculiarities that may throw some light on a whole series of phenomena, such as genome organization, frameshift regulation, protein folding and association, and protein stabilization. Thus, there are a variety of reasons for detailed study of their biochemical and physicochemical properties. The current status of our ongoing studies is described below. A.

Triose-Phosphate Isinnerase

The catalytic role of triose-phosphate isomerase (TIM, EC 5.3.1.1) in glycolysis is to channel all six carbon atoms of fructose 1,Cbisphosphate into the energygaining steps of the pathway by converting dihydroxyacetone phosphate (DHAP) to glyceraldehyde %phosphate (GAP). Kinetic analysis of the reaction of different TIM homologs has shown that at physiological triose phosphate concentrations the catalysis of the enzyme is diffusioncontrolled (Blacklow et al., 1988). All TIM enzymes isolated so far are onedomain (a/3)* barrel homodimers with a subunit molecular mass of 26-27 kDa (Banner et al., 1975). Monomeric TIM constructs retain exceedingly low catalytic activity (Borchert et al., 1994). The TIM barrel is the most frequently occurring folding motif in proteins (Branden, 1991). Comparison of the structures of TIM enzymes from trypanosoma, yeast, and chicken shows that the topology is essentially identical, in spite of the fact that there are 50% sequence differences (Fig. 7) (Wierenga d al., 1992). On the basis of this finding, homology modeling of the known sequences may allow exchanges to be correlated with

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

213

FIG.7. a-Carbon tracings of three superimposed open structures of triose-phosphate isomerase. Thick lines, trypanosomal TIM; thin lines, yeast TIM; dashed lines, chicken TIM. The N and C termini are labeled (Wierenga e l al., 1992).

specific adaptations to extreme conditions (Bdhm and Jaenicke, 1992, 1994). In the case of TIM from 7’. maritima, the tpi gene has been found to be fused downstream to the gene coding for phosphoglycerate kinase, that is, the next but one reaction in the glycolytic pathway (Schurig et al., 1995a). Within the bifunctional polypeptide chain, TIM occupies a length of about 27 kDa at the Gterminal end, which corresponds to the subunit size known for the enzyme from other sources. Comparing all known TIM sequences, a high structural homology to the a/@barrel topology is predicted. Presently, no detailed studies of TIM as a separate enzyme entity are available. Because it represents part of the PGK chain that is expressed both as individual PGK and PGK-TIM fusion protein, we discuss the enzyme in connection with phosphoglycerate kinase (Section V,C).

B.

Glyceraldehyde3-Phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been a paradigm for the structure-function relationship of NADdependent dehydrogenases since the enzyme was first purified and subsequently crystallized and its sequence and threedimensional structure determined (Czok and Bucher, 1960; Buehner et al., 1974; Harris and

214

R. JAENICKE ET AL.

Waters, 1976). Since GAPDH enzymes from various sources (including thermophiles and archaea) have been thoroughly investigated in the past, the T. maritima enzyme was chosen for a comparative analysis of the specific properties of homologs from mesophiles, thermophiles, and hyperthermophiles. The enzyme from yeast, lobster, pig, Bacillus stearothaophilus (Bst), 7'hermus aquaticus ( Tag), and Thmoprotpus tenax (Tst) has been shown to be a homotetramer of about 140,000 molecular weight. It catalyzes the oxidative phosphorylation of the substrate glyceraldehyde-3-phosp hate to I>1,%bisphosphoglycerate, with N AD / NAD H as coenzyme. Interestingly, in Thennoproteus tenax, which is a member of the Crenarchaeota, two GAPDH species have been discovered, one specific for NAD(H), the other specific for NADP(H). The NADP(H)specific enzyme (involved in gluconeogenesis) shows sequence homology and immunological similarity to the GAPDH of the Eitryarchaeota (Hensel et al., 1994). It has been concluded from the conservation of specific amino acid residues and detailed kinetic studies that the catalytic mechanism of GAPDH has been strictly conserved during evolution (for a detailed review, see Harris and Waters, 1976,and Wonacott and Biesecker, 1977). Comparison of the sequences and three-dimensional structures of the enzyme from a mesophile (lobster) and a moderate therniophile (B. stearothophilus) supposedly answered the question of what is the molecular mechanism of thermal stabilization (Harris and Walker, 1977;Argos et al., 1979; Jaenicke, 1981). However, the improved high-resolution structure of Bsl GAPDH failed to confirm the hypothetical additional ion pairs (Skarzynski et al., 1987). Therefore, another approach to the problem, this time using the homolog from a hyperthermophile, seems appropriate. +

I . Wild-TypeEnzyme The GAPDH from T. maritzma has been purified ( Wrba P t nl., 1990a), and its amino acid sequence has been determined at the protein and DNA level (Schultes et al., 1990; Tomschy et al., 1993). Comparing the physicochemical and enzymological properties of Tm GAPDH with those of the enzyme from mesophiles and other thermophiles, no anomalous Arrhenius behavior is detectable over the whole temperature range, except for the slight curvature which is also observed, for example, for the yeast enzyme (Fig. 8A). Discontinuities reported for a number of arc haeal GAPDH enzymes are insignificant and do not seem to be of biological importance (Hensel et al., 1987, 1994; Fabry and Hensel, 1987). Marked differences between Tm GAPDH and mesophilic homologs regarding physicochemical properties are (i) enhanced intrinsic stability, (ii) decreased exchange

-

215

STRUCTURE AND STARII.ITY OF HYPERSTABLE PROTEINS

30

$ 10 a

A

28

32

16

d l l l 1K-'1

0

20 LO Temperature

60

PC)

80

Fic:. 8. Temperature and guanidine (GdmCI) dependence of the specific activity of Tm GAPDH. (A) Activity under standard conditions, in the absence ( 0 ) and presence (0) of 5 m M ATP. Inset: Arrhenius plot. (B) GdmCldependent activation/deactivation (0) and denaturation, monitored by fluorescence emission at 320 nm (A,,, 280 nm) ( A ) and circular dichroism ( 0 ) (Wrba PL nl., 1990a; Rehaber and Jaenicke, 1992).

rates in H-D exchange experiments at room temperature, (iii) activation of the enzyme at low denaturant concentrations (Fig. 8B), and (iv) a relatively small change in hydrodynamic volume on coenzyme binding (Table 111).These properties indicate low protein flexibility, high packing density, and strong subunit interactions in the T. maritamaenzyme at 25°C. At TABLEI11 Properties o j Homologmu Mesophilic and Thermophilic Glycerafdehyde-3-PhosphateDehydropaseP GAPDH Property

T. maritima

Molecular mass (M,) (kDa) Subunit mass (Mi) (kDa) Change in s20,w(holo versus apo)' Thermal transition (7; of holo-GAPDH, "C)' Denaturation transition in GdmCl ( c , , ~2OoC) Relative activation at 0.5 M GdmCl (70°C) Relative H-D exchange rate (& 25°C)" Specific activity (U/mg) & ( pM substrate) & ( p M Nm') Yield of reactivation at O0C/?30"C

145 37 3.5% 109 2.1 M 300% 64 200 (85°C) 400 (60°C) 79 (60°C)

0%/85%

Yeast 144 36 5.3% 40

0.5 M 0% 100 70 (20°C) 160 (25°C) 44 (25°C) 35%/85%

' Data from Hecht ~t al. (1989), Wrba el al. (1990a), Rehaber and Jaenicke (1992). bChangein h,w in the presence of 1 mM NADt, relative to h,w (apo-GAPDH). ' Irreversible denaturation monitored by differential scanning calorimetry. dflml,exchange rate constant according to EX,, mechanism (Hvidt and Nielsen, 1966).

216

R. JAENICKE ETAL.

their respective physiological temperatures, the conformational flexibilities of the yeast and T. maritimu enzymes converge. In shifting from the mesophilic to the thermophilic enzyme, H-D exchange rates result in about 5 kJ/mol for the Gibbs energy of the average local unfolding; the effect may be attributed to the increased saturation of the structure with nonpolar contacts (Wrba et al., 1990a). Considering the amino acid compositions, it is obvious that only the exchanges Lys+ Arg, Ser + Thr, and Val + Ile agree with the previously mentioned traffk rules of thermal adaptation (Section 111,B; Jaenicke, 1994a). The primary structure of Tm GAPDH is highly homologous with those from other less thermophilic bacteria (Schultes el al., 1990). Sequence identities between the enzymes from T. maritima, T h a u s aquaticus, and B. stearothennophilusrange from 59 to 63%, and only 8% of the exchanges are nonconservative. This means that the three homologous GAPDH enzymes, with denaturation temperatures of 110, 80, and 68"C, differ in about one-third of their amino acid sequence, that is, in 100 of 330 residues; which of the differing residues are responsible for the change in thermal stability is obviously not a trivial question (Jaenicke, 1991a). Alignments of the known GAPDH sequences yield one (one-residue) deletion and one (two-residue) insertion (Schultes et al., 1990). Homology modeling results in equal topologies for the mesophilic enzyme and its thermophilic and hyperthermophilic counterparts (BGhm, 1992), in accordance with the observation that their secondary and quaternary structures are highly similar and that the catalytic site residues are conserved in all species. The crystal structure of Tm holo-GAPDH was solved to a resolution of 2.5 A, applying Patterson search techniques on the basis of the molecular model of aSt GAF'DH (Skarzynski d al., 1987; KorndGrfer et al., 1995). The final model is made up of two monomers in the asymmetric unit, with 332 amino acids each. They are related to the other two monomers by a crystallographic 2-fold axis, forming a tetramer with approximate 222 symmetry (Fig. 9A). The overall mean positional error is estimated to be 0.26 A. As expected from the high level of sequence homology,

FIG.9. Structure of T. marilima glyceraldehyde-%phosphate dehydrogenase at 2.5 A resolution. (A) Structure of the tetramer. Secondary structural elements are shown as given by DSSP graphics created with MOLSCRIPT. (B) Stereo view of CF backbones.of the subunit of Tm GAPDH (thick lines), and Brt GAPDH (thin lines) after least-squares superposition of catalytic domains (cf. Korndbrfer .d aL. 1995).

A

0

Q

218

R. JAENICKE ETAL.

the crystal structures of Bst and T m GAPDH are very similar (Fig. 9B). Fitting the catalytic and NAD-binding domains separately, the rms deviation of superimposed C" positions is found to be only 0.57 and 0.83 respectively. The insertions and deletions are found in loops on the surface of the molecules. The overall rms deviation for the entire monomer is much higher, 2.56 owing to a rigid body rotation of 4.4" of the two domains relative to one another. The Gterminal helix (residues 312-331) belongs structurally to the NAD-binding domain and is connected to this domain through salt bridges. In determining local differences between the known threedimensional structures, it seems useful to include the lobster structure (Buehner et al., 1974) because this extends the physiological temperature range under study from 90°C down to about 20°C. As summarized in Table IV,the pattern of peripheral ion pairs shows significant differences in the three enzymes: the total number in Tm GAPDH is substantially higher than in the B. stearothnmophilus and lobster enzymes, whereas the number of intersubunit ion pairs is reduced (for detailed discussion, see Kornddrfer et al., 1995). Ionic interactions of residues in contact with Arg-10 form a complex charge cluster that is conserved in all GAPDH structures, suggesting an essential role in the stabilization of the native state of the enzyme (see Section V,B,2). Considering the mechanism of thermal stabilkation, Bst and 7'm GAPDH, as well as the enzyme from lobster, are closely related and should, therefore, be a good paradigm to evaluate the traffic rules proposed by Argos et al. (1979). In extending this work, MenCndez-Arias and Argos (1989) used 70 different sequences, 18 of them representing GAPDH enzymes. Given this large sample and the above-mentioned high sequence homology, one would predict that 7 m GAPDH fits well into these models. Most frequent exchanges from Bst to 7'm GAPDH are Gly + Ma, Ser -+ Thr, and Ile + Val (numbers 3, 4, arid 5 in the statistics). The Lys -+ Arg and Ser + Ala exchanges (rank 1 and 2) occur only twice and once, respectively. Instead, there are four Phe -+ Leu exchanges, which do not occur at all within the top 10 exchanges. Also the prediction that thermal adaptation increases the Ala content of a helices is not confirmed by Tm GAPDH as this contains 7 Ma residues less than lobster GAPDH, and 14 less than Bsl GAPDH. N o preference for substitutions with residues of higher helix propensity is observed, nor is there a noticeable preference for a-helical positions in the changes toward Ma from mesophiles to the hyperthermophile. In summary, in the case of T m GAPDH, the predictive power of the traffic rules is limited. Ishikawa et al. (1993) noted that the local structure of each mutated site must be carefully inspected in order t o understand

A,

A,

219

STRUCXURE AND STABILITY OF HYPERSTABLE PROTEINS

TABLEN Selerted Peri$heral (Intrasubunit) Ion Pairs in Glyceraldehyde3$1hosflhateDehydrogenase" Residue 1

Residue 2

Arg-10 Arg-10 Arg-20 Arg-20 Lys-56 Lys-81 LyS-81 Lys-101 Arg-102 Arg-102 Lys-104 Lys-107 Lys-107 Lys-114 Lys-136 Lys-159 His-190 Arg-195 Arg-195 Arg-197 Arg-245 Lys-256 Lys260 Lys-260 Arg-266 Arg-288 Lys-303 Total

Glu-314 Asp47 Asp323 Asp326 Glu-58 Glu-76 Asp78 Glu-103 GIu-106 Asp125 Glu-103 Asp-78 Asp104 Asp90 Asp1 35 Glu-I63 Asp181 Asp181 Asp192 Asp186
Bst

HA

++++ ++++ -

-

-

++++ ++++

-

+(+++)

-

++++

-

-

-

+++(+) ++(++) +(+)

-

+++(+)

++++

+(++) +(+++) +(+++)

-

++++

55(17)

++++ ++++ ++++ ++++ ++++ -

(+++I

++++

-

(++)

59(33)

Tm

++++ ++++ ++++ ++++ ++ ++(++) ++++ ++++ ++++ ++(++) ++(++)

-

++(++)

-

++(++)

++++ ++++

(++++)

++++ -

(++)

++++ ++++ -

(++)

73(32)

From IImnarus ama'canus ( H A ) , R. sttarothaophilus (Bst), and T. rnaritima ( 7 r n ) . Residue numbering and residue types have been taken from Tm GAPDH, except where no salt bridge at all is found in Tm GAPDH. For the corresponding residue numbers and residue types for H A and Bst, see the Brookhaven Protein Data Bank. Only ion pairs up to a distance of 4.0 A are included. + stands for an ion air in each of the four chains in the tetramer. ( + ) stands for an ion pair within up to 6.0 that failed the 4.0 A distance criterion but has corresponding pairs in either another subunit or one of the other enzymes shown here

1

effects on the properties of proteins. As they found out, Gly residues may cause destabilization by an increase in chain entropy of the unfolded state; on the other hand, Gly residues can stabilize by release of steric hindrance. To illustrate this, Val-237 in Bst GAPDH has (+,$) values of ( - 114",123") and lies outside the allowed range for nonglycine residues. The conformational angles of Gly-237 are (114",127");thus, the substitu-

220

R. JAENICKE ETAL.

tion Val + Gly in Tm GAPDH may contribute to the higher thermostability of this enzyme. A significant stabilizing effect seems to be attributable to the increased number of intramolecular ion pairs. Perutz and Raidt (1975) suggested that thermal stabilization of proteins can be provided by salt bridges on the molecular surface rather than in the inner core of the molecule. The structure of Tm GAPDH confirms this hypothesis. With one excep tion (Asp323),all ionic bonds that are buried are conserved in all GAPDH structures (Kornddrfer et al., 1995). All additional ion pairs attributable to the thermostability of Tm GAPDH are located in the periphery of the tetramer. In contrast to observations on other oligomeric proteins that intersubunit ion pairs are essential for thermal stabilization (Perutz and Raidt, 1975), their number is decreased in Tm GAPDH (Table V). This results in a charge pattern for Tm GAPDH that is more similar to that of the lobster enzyme than that of Bst GAPDH, indicating TABLE V

Selecied Intersubunit Ion Pain i n Glymal&hyde3-Phmphate Dehydmgenase" Residue 1

0 - P interface 4 - 1 6 9 (o/p) Arg-194 ( 0 ) Arg-194 ( 0 ) 4 - 1 9 7 (o/q) Arg-169 (p/r) Arg-169 (p/r) Arg-194 ( p / d Arg-197 (p/r) 0-Q interface Lys-45 W p ) Lys-45 (q/r) 0 - R interface Arg-10 (o/p) Arg-10 (9) Arg-10 (r) M3-281 Total

Residue 2

HA

Bst

7m

Asp245 (p/r) Asp277 (p) Asp293 (p) Asp282 ( o h ) GIU-245 (O/q) Asp301 (o/q) Asp293 (o/q) Asp282 (o/q)

(+)

+

-

+ +

+ + + + + +

-

Glu-276 (q/r) Glu-276 (o/P)

(+I

(+I

Asp186 (r/q) hP-186 (p) Asp186 ( 0 ) Glu-201 (q/p)

+ +

+

(+)

-

+ + + + + + +

13(11)

23(5)

16(0)

+ + +

(+)

(+)

-

-

+ + +

+ + -

-

-

'From Homarus amen'canus ( H A ) , B. stcorothmnophilus ( t h t ) , and T. maritima (I'm). Residue numbering and residue types have been taken from Tin GAPDH, except where n o salt bridge at all is found in Tm GAPDH. For the corresponding residue numbers and residue types for HA and Bsf, see the Brookhaven Protein Data Bank. Only ion pairs up to a distance of 4.0 A are included. + stands for an ion pair within up to 6.0 A that failed the 4.0 A distance criterion but has corresponding pairs in either another subunit or one of the other enzymes shown here.

22 1

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

that not all intersubunit ion pairs are important for the thermal stability of oligomeric proteins. Another factor contributing to the anomalous intrinsic stability of hyperthermophilic proteins may be the compactness of the molecule, caused by increased van der Waals interactions (Privalov and Gill, 1989). In connection with Tm GAPDH three independent findings point to increased packing: decreased rates of H-D exchange, the anomalously small effect of NAD' binding on the hydrodynamic volume, and the 3fold activation of the enzyme at low concentrations of guanidinium chloride (Fig. 8B, Table 111). Comparison of the accessible surface area and volume from X-ray data, as a possible measure for compactness in the case of the different GAPDH enzymes, does not correlate with the thermal stabilities for either the monomer or the tetrarner (KorndBrfer et al., 1995). The loss of ionic intersubunit interactions is reflected in a decreasing surface area of charged residues buried in the tetramer contacts on going to more thermophilic proteins (Table VI).At the same time the surface area of hydrophobic residues buried in the tetramer contacts increases with thermostability. This means that increased hydrophobic interactions between the subunits may lead to a stabilization of the tetramer with respect to subunit dissociation. 2. Recombinant Enzyme, Mutants, and Constructs The previous discussion of possible strategies of thermal adaptation suggests recombinant DNA techniques as the method of choice to prove or disprove hypothetical mechanisms. In cloning T m GAPDH there were two aims apart from large-scale purification and sitedirected or random mutations. First the production of a hyperthermophilic protein in a TABLE VI Accessible Sutjace Areas and Buried Areas in Tetramer Contacts of GlyceraMehydP-3-PhosphateDehydrogenasef' Accessible surface charged residues bured in tetramer contacts

Genus and species

Accessible surface area buried in tetramer contacts (A2)

Accessible surface area of hydrophobic residues buried in tetramer contacts (A2)

Homarus americanus B. s&arothermophilus T. niuritima

13.807 15,543 14,641

5789 7551 7628

3220 5028 3693

(A2)

" Ala, Ile, Leu, Met, Phe, Pro, Trp, and Val were considered to be hydrophobic residues; Asp, Arg, Glu, His, and Lys were considered to be charged residues.

222

R. JAENICKE ET At..

mesophilic host, such as E. coli, allows in viuofolding experinients under conditions very different from the thermophilic conditions in sztu. Second, using deletion mutants or other modifications of the 7’m GAPDH gene, constructs of the enzyme can be designed to address questions regarding the stability and folding of domains, as well as their interactions in the native tertiary and quaternary structure Uaenicke, lYY4a). The cloning, expression, and purification of T m GAPDH have been described (Tomschy et al., 1993). In designing the oligonucleotide priniers, the codon usage of the known genes of 1 . mantima was taken into account. It is important to note the fact that the semisynthetic gene obtained from the polymerase chain reaction and the synthetic linkers coding for the lacking N- and Cterminal gene segments were in complete agreement with the amino acid sequence determined by fragmentation and Edman degradation (Schultes et al., 1990) (Fig. 10). A gupdh- strain was used for the expression of the recombinant gene in li. coli. Because expression of the recombinant enzyme was found to restore the common phenotype of E. coli, the hyperthermophilic enzyme must he expressed in a form active enough to support growth at a teniperatiire more than 50°C below the normal in viuo conditions. Considering the marginal free energy of globular proteins, the low sensitivity of folding and association of the enzyme with respect to temperature is rather unexpected. It is confirmed by temperature-dependent reconstitution experiments (after prior denaturation with guanidine), as these yield up to 85% of the native enzyme at temperatures between 10 and 90°C (Kehaber and Jaenicke, 1992). Physicochemical studies show that the recombinant enzyme is identical to the native form of Tm GAPDH in hydrodynamic, spectral, and denaturation/renaturation properties. The same holds for the enzymological properties: the specific activities (46 U/mg), the kinetic parameters for the catalyzed forward reaction (Km,NAD+ 12 p M ) , and the inhibition by glyceraldehyde %phosphate ( 21 mM) or arsenate (>10 mM) are iden tical within the limits of error (Tomschy et aL, 1993). In addition, the crystal data collected for the natural and recombinant proteins were identical. The design of Tm GAPDH has focused mainly on obtaiiiing temperature-sensitive forms of the enzyme, for the simple reason that spectroscopic and thermodynamic measurements above 100°C lead to unsurmountable practical problems. On the basis of a calculated model for the structure of Tm GAPDH (Bdhm, 1992) and preliminary X-ray data, Tomschy et al. (1994) directed the selection of sites for niutagenesis toward ion pairs that are present in Tm GAPDH, but not in Bsl GAPDH. Following the above arguments and the prediction by Peruu and Raidt (1975), removal of such salt bridges might shift the limit of stability

I

I

70

ARVAlNGfGRlGRLVVRl IVfRKNWIEVVAlNDLI~DlKTLAHLLKVDSV~KfPGKVEVlfNSL IVDG Kf IKVfAEPDPSKLPYKDLGVDFVlfSlGVFRNRfKAELHLQAGAKKVlIIAPAKGEDIlVVIG~NfDQLKP.tHII I S C A S C I I N S I A P I V K ~ ~ ~ ~

2 IVKVG.. . . . .. . .N.F .AAL- KNPDlf.VAV.. LT.NMGLWLL.. mVG.. . . .. . . 4

140

1.1- HSRGVE

. .VHGRLDAE .VVNffiDVSVN. , f . IVKAERN.tNLA.GEIGVDIVVE.. .

1 . .LT-NDKTLAHLL. . . IVHRf PGE .AVDWVLVVD. .A. RA1AVKD.KflP.AEAGVGVW If.

.R.lKRED.AK.LEA.. .. . I . S . .AKVfNIIV.M.VNQDKV DpIAHHVl .N.. . . . . c ~.FA. . .LHQ

. .V. 1DAM.KA. LEG. . . . . I. I. .AKGEDllL .M.VNHEAV DPSRHHI I .N.. . . . .S L , .m. ,LEE

VRVA.. . . , - .. .L.m.IAL--SRFWE.WAS. .PfIBLDVAAVMF.. . .lHGRVAGE.SHDDKHIIVD. .K.AIV~ERD.ANLP.SSGDSWIAID...V.KtLDl.QK. IDA.. . . . V . l . . S - S l A W ~ . M . V D S ~ K V ~ - S D L K l ~ .. .N...CL. . .LA.. lm

5 ACSKlG..

.... ... L . L . M L - - SCG-AQ.VAV..PFlAL~VMVVMF ....1HGVfKGE.KMDCALVVD. .K.IVFNEM.ENIP.SKAGA~VIVE...V.IIIEK.SA.fKG.. . . . V . S . 210

.S-ADAFUf.C.VNLEKVS-KCMlVV.N..

. _..CL..VA..LHE

280

1 KFGlVSUllTlYHSVTWOPRVLDLP-~DLRRLRAPIIVNIlPlITGAAKAVALVVPEWKGKLD~IRVPIPffiSIIDLIVLVEKEIIVfEVMVfAIfGRLKGI IGVNDtPlWSSDl IGllfSGIFDAlIlN~lGGKLWKVASUV~EVGVSNRVVDlLfLLL~

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FIG.10. Amino acid sequences of glyceraldehyde-%phosphatedehydrogenases from ( 1 ) T. mantima, (2) B. steamthmnuphilw, ( 3 ) T h w (4) Sacchuronzyces cembiae, and (5) Homam ammicanus (lobster). Numbering refers to the T. maritima sequence; dots (.) represent identical amino acids, and dashes (-) denote deletions (cf. Schultes el al.. 1990). aquaticus,

224

R. JAENICKE ETAL.

from the hyperthermophilic to the moderately thermophilic temperature range. In choosing potential candidates, three categories of ion pairs in different parts of the subunits of Tm GAPDH were selected: (1) nonconserved charges in Tm GAPDH that are not present in Bst GAPDH, (2) highly conserved salt bridges involved in the Arg-10 charge cluster in the N-terminal (NAD-binding) domain, and (3) ion pairs in the Sloop of the substrate-binding domain, which is involved in tertiary and quaternary contacts in the enzyme. All three types of mutation sites are located in the periphery of the tetrameric enzyme (see Tomschy et al., 1994). The first category included G l ~ ’ ’ ~ - L y-+s ~Gl~i~~’-Asp“’~ ~ and G1uZ6’changes. These involve peripheral residues remote Arg2ffi G1uZ6’-G1um from the active site and not engaged in subunit contacts. They were found to have no effect on enzyme activity and quaternary structure. In addition, the thermal stability of the mutant proteins was unchanged, suggesting that the stabilizing effect of peripheral ion pairs depends o n the structural context and cannot be generalized. In the second case, the Arg-10 charge cluster involves at the same time two intrachain and one intersubunit ion pairs; in addition, the backbone imino group forms one out of eight hydrogen bonds responsible for coenzyme binding. Replacing Arg-10 by Met or Lys (to keep the backbone in place and to explore how critical the charge distances are) led to the uncoupling of guanidine deactivation and denaturation. Deactivation of both mutants occurred at exceedingly low denaturant concentrations, reflecting the release of the coenzyme due to preferentially local structural changes. On the other hand, denaturation, as monitored by the change in fluorescence emission, remained unaltered, proving that the mutations have no significant effect on overall protein stability. The third case involved the Sloop (residues 178-201) which in mesophilic GAPDH enzymes is highly conselved. In the thermophilic enzymes, there is a unique ion pair, A~p’~’-Arg’~~, which in the case of Bst GAPDH has been suggested to play a role in the stabilization of the tertiary and quaternary structure (Skarzynski et al., 1987). A single point mutation, Argl95Asp, and a double mutant, Aspl81Lys-Arg195Asp, were constructed to test this hypothesis. The latter was designed as a suppressor mutant where, for steric reasons, Arg was replaced by Lys. Both mutants showed significantly reduced specific activity, owing to the involvement of Arg-195 in the binding of the “substrate” phosphate ion. There was also a decrease in stability as well as a change in cooperativity, but there were no changes in the tetramer-monomer transition, even at low temperature and concentrations below 0.1 yM. In summary, changes in peripheral ion pairs cannot be considered a staightforward strategy of thermal adaptation. Even after carefully select-

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

225

ing specific loci for site-directed mutagenesis on the basis of multiple sequence alignment and homology modeling, no predictable results have been obtained. Instead, each of the mutations unveils new and unexpected properties of the oligomeric protein. Recalling that the overall stability of proteins is a minute difference between large contributions of attractive and repulsive forces and the corresponding entropy contributions, this result is not too surprising. As has been mentioned in connection with the hierarchical structure of proteins and the contributions of domains and/or subunits to the overall stability (Section II,B), significant AGsrabincrements may come from domains and domain interactions. In the case of lactate dehydrogenase (which is topologically related to GAPDH) , fragmentation studies have clearly shown that there is a continuous decrease in stability in going from the tetramer to the dimer, monomer, and, finally, to domains (Opitz et al., 1987). Another model case involves eye lens crystallins, where limited proteolysis and cloning of separate domains of yB-crystallin have shown that the interdomain contact is essential for the anomalous stability of the protein (Mayr et al., 1994). In the present context, one may ask whether in the case of hyperthermophilic proteins, such as Tm GAPDH, compact substructures or domains are independent folding units showing anomalous intrinsic stability. Using the coenzymebinding domain of the enzyme as an example, it was shown that the substructure binds NAD' and NADH with high affinity and possesses the same guanidine unfolding transition as the native tetramer ( c ~ ,=~ 2.2 M ) , indicating (i) that the excised domain is folded as in the wildtype tetramer and (ii) that the constituent parts of the molecule share the high overall stability of the parent molecule (Jecht et al., 1994). The construct underlying this study was the most conservative variant, simulating the native combination of the compact N-terminal domain (residues 1-146) with the C-terminal ashelix (residues 313-333). It would be interesting to know how the ashelix is involved in the stabilization of the core domain (residues 1-146). This contains the Rossmann fold common to many dehydrogenases. Obviously, during protein synthesis its interaction with the C-terminal domain must wait until the entire polypeptide chain has been synthesized. Because monomer folding and oligomerization are coupled processes, it is conceivable that locking the domain by means of its Gterminal helix is an important step in the assembly of the enzyme.

3. Stability and Folding The in vitro reconstitution of glyceraldehyde-3-phosphate dehydrogenase after preceding denaturation and dissociation obeys the sequential uni-bimolecular ( Uni-Bi) mechanism according to

226

R. JAENICKE ETAL.

(Rudolph et al., 1977; Rehaber and Jaenicke, 1992). A kinetic scheme with consecutive folding and association steps is what one would expect for any oligomeric system, because complementary recognition sites are a necessary prerequisite for proper docking. At high enzyme concentrations, kinetic partitioning leads to aggregation rather than tetramer formation (Jaenicke, 1987, 1996). In the case of mesophilic GAPDH (from yeast), two observations are of special interest in connection with folding and association. First, ATPdependent cold inactivation yields nativelike monomers still capable of NAD' binding. Their thermal reconstitution again obeys Eq. (3),proving that late events in the reconstitution pathway must give rise to ratedetermining folding steps in the overall process of structure formation (Bartholmes andJaenicke, 1978).Second, the coenzyme NAD' not only stabilizes the enzyme but, at the same time, enhances the folding rate of GAPDH by shifting equilibria between early intermediates in the folding pathway toward the native tetramer (Krebs et al., 1979;Jaenicke et al., 1980). The GAPDH from T. maritima differs from the yeast enzyme in several respects: (1) there is n o detectable cold inactivation in the absence and in the presence of ATP; (2) the apo- and holoenzyme differ only marginally in thermal stability (Fig. 11); and (3) the rate and yield of reactivation show a complex temperature dependence. For example, using conditions of maximum long-term stability, reconstitution over a wide temperature range (30-85°C) yields approximately 85% native enzyme. At O"C, even after long incubation (10 days), no significant regain of activity is detectable; at temperatures beyond 85"C, thermal denaturation of folding intermediates competes with refolding, still leaving 30% reactivation at 100°C (Fig. 12). Evidently, thermal adaptation of the hyperthermophilic enzyme refers not only to the native tetramer but also to the intermediates in their different states of association. Such intermediates are not explicitly included in Eq. (3);however, it is obvious that there must be at least dimers on the association pathway, because tetramer formation can only occur as dimerization of dimers. The kinetic scheme also does not consider that rate-determining isomerization reactions may occur at the level of the final state of association, giving rise to an apparent unimolecular reaction. To illustrate this alternative, Bst pyruvate dehydrogenase may serve as an example. The reconstitution of the 10-MDa multienzyme complex obeys first-cwcler kinetics, suggesting that the bimolecular association reactions of the 240-mer are fast compared to the folding and/or shuming at the level of the macroassembly (Jaenicke and Perham, 1982).

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

227

75 -

Temperature "C

60

80

Temperature ("C)

lo0

FIG.1 1 . Thermostability of a p e (open circles) and hologlyceraldehyde-%phosphate dehydrogenase (closed circles in the presence of equimolar concentration of NAD' ( 0 ) and 0.2 mM NADt ( O ) ,respectively, from T. man'lima in 50 m M phosphate buffer, pH 6.0, 1 mM dithioerythritol, and 1 m M EDTA. Activity measurements were made under standard conditions of the assay in the presence of 1 pM trypsin. Inset: Thermal analysis using differential scanning calorimetry: solid line, apoenzyme; dashed line, holoenzyme in the presence of excess NADt (cf. Wrba el al., 1990a; Rehaber and Jaenicke, 1992).

A

B .

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I

h

8 .E > .c.

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2

8

v

2 a 50

50

s

0

0

0

60

120

Time (h)

180

m

0 20 40 60 80 100

Temperature ("C)

FIG. 12. Effect of temperature on the reactivation of T. maritimu glyceraldehyde-3phosphate dehydrogenase after preceding denaturation in 6 M GdmCI. (A) Time course at 5°C ( O ) , 10°C ( O ) , 20°C (V),25°C ( 7 ) .40°C (a),and 80°C (m), obtained under standard test conditions. Relative activity refers to final yields after 4 days. (B) Effect of temperature on the final yield of reactivation (Rehaber and Jaenicke, 1992).

228

R. JAENICKE ETAL

In the case of Tm GAF’DH, the transition from the uni-bimolecular to the unimolecular mechanism is detectable in the concentration range of common renaturation experiments. Reactivation of the guanidinedenatured enzyme depends on protein concentration only at extremely high dilution (<4 pg/ml) ; beyond this limit association becomes diffusion controlled, so that the overall reaction is exclusively determined by the folding of the monomer. Because reactivation kinetics monitor only those enzyme molecules that are accessible to reconstitution, irreversible side reactions do not interfere with a quantitative analysis.Assuming the tetrameric enzyme to be the only active species, the reactivation kinetics may be quantitatively described by one single pair of first- and second-order rate constants [cf. Eq. (3) and Rehaber and Jaenicke, 19921:kl = 2.3 X sec-’ (for the folding reaction) and k2 = 8.3 X lo5 liter mol-’ sec-’ (for association). The good agreement of the observed and calculated data may be taken to confirm the mechanism, especially with respect to the assumption that intermediates in the pathway of reconstitution do not contribute to catalytic activity. Bearing in mind that pairs of subunits in GAF’DH share the active sites, this is not trivial. Considering the temperature profile of reactivation (Fig. 12B) and the fact that there is no cold denaturation detectable, the question arises as to the physical nature of the “cold intermediate” (Ioa(:) trapped in reactivation experiments at 0°C. As mentioned, the intermediate is stable over days so that its characterization is feasible. Temperature shifts show that the enzyme regains its active (and fully native) state within manual mixing time (Fig. 13A), thus proving that the zero level of reactivation is attributable to the formation of an inactive intermediate in the reconstitution pathway rather than irreversible side reactions. Ultracentrifugal analysis of the trapped intermediate at 0°C yields a nonlinear In cversus ? dependence corresponding to a weight-average molecular mass of 110 kDa, with the 36,000 monomer as low molecular weight species. Shifting the temperature (within the same experiment) back to 25°C leads to the homogeneous 144-kDa tetramer. Monitoring the kinetics of the Ivc +N reconstitution, fluorescence emission and circular dichroism show qualitatively different profiles. In fluorescence, the biphasic overshoot reaction reflects the fast collapse of the hydrophobic core (with aromatic residues still exposed to the solvent) and subsequent shuffling (still at an enhanced level of fluorescence). Shifting the temperature to 50°C leads back to the native state (Fig. 13B). The change in circular dichroism at 222 nm parallels the fast collapse, leading to nativelike secondary structure. It does not show any significant change when the temperature is shifted to 50°C (Fig. 13C). Thus, there are no significant

50°C

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a

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1

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Time (h) FIG. 13. Cold intermediate on the folding path of T. mon'tima glyceraldehyde-% phosphate dehydrogenase at 0°C. Renaturation was done in standard phosphate buffer, pH 7.0, after preceding denaturation in 6 M GdmCI. After final yields were reached (arrows), the temperature in the 0°C samples was shifted to 50°C. (A) Reactivation at 0°C ( 0 ) and 50°C ( 0 ) . (B) Recovery of native fluorescence at 320 nm (Aem 280 nm) in phosphate ( A ) and Tris-HCI buffer, pH 7.0 (A). Arrows mark temperature shifts to 50 and 3°C. Relative fluorescence of the native control was set to 100%. (C) Recovery of native dichroic absorption at 222 nm. Amplitude at 222 nm for the native enzyme a t 40°C was set as 100% (Rehaber and Jaenicke, 1993).

230

R. JAENICKE ETAL.

changes in helicity during the transition from the trapped intermediate to the native state. On the other hand, the near-UV circular dichroism clearly points to alterations of tertiary contacts in the environment of aromatic residues (Schultes and Jaenicke, 1 9 9 1 ) . Including the sedimentation data, all criteria clearly show that the cold intermediate represents an “assembled molten globule” with the secondary structure close to the native state but the aromatic residues still exposed to the aqueous solvent. However, in contrast to the canonical molten globule, the cold intermediate shows highly cooperative denaturation transitions, with characteristics close to the native enlyme at room temperature. The differences in the limiting guanidine concentrations for the fluorescence and far-UV circular dichroism denaturation profiles clearly indicate that full exposure of the fluorophores precedes the helix-coil transition (Rehaber and Jaenicke, 1993).

C. Phosphoglycerate Kinase and Phosphoglycerate Kinase- Triose-Phosphate Isomerase Fusion Prota’n

1. Homologous Phosphoglycerate Kinases and Fusion Protein Phosphoglycerate kinase (PGK, EC 2.7.2.3) catalyzes the energy-pro ducing first substrate level phosphoIylation in glycolysis, transforming 1 ,Sbisphosphoglycerate to Sphosphoglycerate and, at the same time, transferring the phosphoryl group from the C1 position of the substrate to the y position of ADP. The enzyme is ubiquitous and shows high structural similarity when homologs from various sources, including mesophiles and thermophiles, are compared (Scopes, 1973; Watson and Littlechild, 1990;Joao and Williams, 1993). Since the enzyme is monomeric and contains neither disulfide bonds nor cofactors, it seems well suited as a paradigm for the analysis of strategies of thermal adaptation of proteins. The protein has a molecular mass of about 45 kDa and folds into two distinct domains of approximately equal size. The active site lies deep in the cleft between the two lobes. Movement of the two lobes relative to one another seems to be an essential element in the catalytic mechanism (Banks et al., 1979). The high-resolution structures of the enzymes from yeast and B. stearothennophilus have been determined (Fig. 14) (Banks et al., 1979;Davies et al., 1993).Characteristic features of thermal stabilization include increased hydrophobicity of the core of the protein, additional ion pairs, and enhanced helicity. Thermal analysis and denaturation/renaturation studies of the yeast enzyme showed that the domains differ in their stability and fold indepen-

STRUCTURE AND STABII.ITY OF HYPERSTABLE PROTEINS

23 1

FIG.14. Schematic representation of the open form of yeast phosphoglycerate kinase, viewed from the solvent looking toward the substrate-binding region. The binding sites for Mg-ATP and 3 P G lie behind helices W/IX and helix XIII, respectively. Helices are denoted by cylinders, sheets by arrows. The thick solid line represents the major deletion (amino acids 128 to 142) that is found in eukaryotes but not in prokaryotes.

dently; the Cterminal domain is found to be more stable than the Nterminal one (Burgess and Pain, 1977; Adams et al., 1985; Betton et al., 1989). Irreversible heat aggregation does not allow the thermodynamic parameters of thermal unfolding to be determined in a straightforward manner (Galisteo el al., 1991). Semiquantitative data were obtained for the enzyme from yeast and Thermus thermaphilus,where it was shown that thermal adaptation leads to a flattening of the AG,,, versus temperature parabola rather than to a shift to higher temperatures (cf. Fig. 2B) (Nojima et al., 1977, 1978, 1979; Oshima, 1979). The molecular basis for these characteristics is still unknown. On screening for PGK activity in crude extracts of T. man'tima grown under standard conditions, two well-separated peaks with PGK activity are eluted on gel permeation chromatography (Schurig et al., 1995a). Calibration with standard proteins reveals molecular masses of around 45 and 350 kDa. The relative amounts of the two species are the same. Rechromatography and purification of each of the two fractions show that there is no conversion of the two enzyme species. Thus the two activity peaks d o not belong to one and the same enzyme in different states of oligomerization. As taken from the range of molecular weights

232

R. JAENICKE ETAL.

of known PGKs, the low molecular weight species may be attributed to “normal” PGK, in accordance with its N-terminal sequence. In analyzing the “large” PGK, it turns out that in each purification step TIM activity coelutes with the PGK activity. In the pure 350-kDa fractions, the TIM activity is found to exceed 500 U/mg (at 40°C), which clearly proves that the TIM activity cannot be attributed to an impurity. Using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, no protein band is obtained in the 25-30 kDa range where TIMs from other sources would be typically found. Furthermore, protein sequencing reveals the same N-terminal sequence observed for the low molecular weight PGK Consequently, the large PGK must be a covalent PGK-TIM fusion product with TIM as the Cterminal part of the bifunctional enzyme. The complete sequence of the corresponding gene cluster corroborates this conclusion (Schurig et al., 1995a) (see below). Physicochemical characterization of PGK and the PGK-TIM fusion protein is summarized in Table VII. As judged from gel-permeation TABLE MI Physicochical and Catalytic CharacrcriLation of Pharphoglyccrale Kinase and PGK-TIM Fusion tfolein jEom Thennotoga m a r i t i d Characteristic Molecular mass (kDa) M, (calculated) MI (SDSPAGE) U, (ultracentrifugation) K O(UC/dynamic light scattering) M (gel filtration) Sedimentation constant (sqg.*) Diffusion coefficient (&,J Absorbance A*,lm (O.l%)b Enzymatic properties Specific activity (U/mg. 40°C) pH optimum of activity &rPp (40°C) Activation energy‘

Thermal transition (Tm)

Tm PGK-TIM fusion protein

Tm PGK

71.6

43.2 43 2 3 45 2 4

71 2 3

260 2 20 286 2 9 370 2 SO 8.6 & 0.3 X lo-”

-

sec

2.7 X lo-’cm4 sec-’ 0.56 2 0.03 200 (PGK activity) 500 (TIM activity) 5.2-6.0 (PGK) 7.5-9.0 (TIM) 0.7 m M (3PG)

37 kJ/mol (PGK) 80 kJ/mol (TIM) 100 “C (PGK) 88OC (TIM)

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-

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5.2-6.0 0.3 m M (SPG) 38 k]/mol

100°C

SDSPAGE, SDS-polyacrylamide gel electrophoresis; diffusion coefficient from dynamic light scattering (M. Ott, unpublished 1996; Schurig ef al., 1995a). Determined according to Gill and von Hippel (1969). ‘Activation energy of enzyme catalysis at 20-70°C (Arrhenius).

’

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

233

chromatography, SDS-polyacrylamide gel electrophoresis, sedimentation analysis, and dynamic light scattering, the two proteins are homogeneous. The molecular mass of the monofunctional enzyme is in agreement with all other known PGKs reported so far. In contrast, the PGK-TIM fusion protein is a homogeneous stable tetramer. In the ultracentrifuge, even under conditions of meniscus depletion, no significant dissociation to dimers or monomers can be detected. The significant difference in the molecular mass derived from size exclusion chromatography (370 kDa) and sedimentation equilibrium (260280 kDa) may reflect an anomalously large hydrodynamic volume or asymmetry of the tetramer, which is also suggested by dynamic light scattering measurements. The combination of the sedimentation and diffusion data precisely confirms the tetrameric quaternary structure: M,D= 286 kDa. Given the molecular mass of the subunits, the difference between the fusion protein, 70 kDa, and normal PGK, 43 kDa, corresponds well to the molecular mass of a TIM subunit from a wide variety of species (26 t 1 kDa). Generally, TIM enzymes are homodimers held together by an interfacial loop located opposite to the N terminus (Wierenga et al., 1992). Several residues that fold into this loop are found to be conserved in the Tm TIM primary structure. Assuming the common TIM topology to hold also for the Tm enzyme, a linkage of the N terminus of TIM to the C terminus of PGKwould allow the normal dimerization of the isomerase. The stabilizing forces that favor dimerization of the dimer are unknown. 2. pgk and tpi Genes The previous findings regarding the fusion of TIM to the C terminus of PGK are also reflected by the organization of the corresponding gene cluster of T. man'tima. At the DNA level, the gap gene (which encodes GAT'DH; Tomschy et al., 1993) is followed by the pgk and tpi genes (encoding PGK and TIM). The observed gene cluster resembles the operon of several bacteria, such as the gap operons of Bacillus megata'um (Schliipfer and Zuber, 1992) and Corynebacta'um glutamicum (Eikmanns, 1992). As deduced from the DNA sequence, the 1200 bp (including the stop codon) of the pgk gene encodes a 399-residue protein with a calculated molecular mass of 43,190 Da (for the amino acid sequence, see the PGK sequence alignment in Fig. 16). The following tpi gene lacks a start codon, as well as promoter elements and the ribosomal binding site. Obviously, tpi must be expressed together with the preceding pgk gene and, for this reason, is not present as a distinct enzyme in T. maritima. Moreover, both pgk and tpi are not fused in frame. The pgk gene ends with a stop codon that overlaps the TIM codon sequence. The fusion

234

R. JAENICKE ETAL

protein shares the N-terminal domain of 399 amino acids with PGK, followed by approximately 255 amino acids of the T I M sequence. A mechanism such as a programmed reading frameshift must be in effect upstream of the PGK stop codon. Such natural subversion of the triplet decoding on translation is involved in the expression of several prokaryotic and eukaryotic genes (Levin et al., 1993; Farabaugh et al., 1993). Studies on the mechanisms led to the conclusion that all programmed frameshifts depend on (i) a "recoding site" that allows nontriplet slippage of the mRNA and (ii) a stimulator that increases the efficiency of recoding. Among other mRNA characteristics, secondary structure elements and rare codons may stimulate the frameshift events by causing translational pauses. Depending on such signals, translational frameshifts yield efficiencies o f 50% and more (Schurig et al., 1995a). With the !us gene (i.e., the pgh and tpi gene including the frameshift), no secondary structure elements or ribosome binding site is detectable, but the poly(A) sequence may act as a slippage site in combination with the in-frame stop codon (Fig. 15). t.:scha'chia coli RNA polymerase was found to slip on 10- to 1I-nucleotide-long runs of A or T bases (Wagner et al., 1990). If we assume in the present case that a - 1 negative frameshift upstream of the stop codon is in effect to link the pgk and tpi genes (so that the linker might be . . . IKKKITRKL . . . ), the 1964bp DNA sequence of the fus gene (including the stop codon) encodes a 654residue protein with a calculated mass of 71,600 Da. Some of the previously mentioned genes have been expressed in E. coli. The pgk gene yields an enzyme that is indistinguishable from native T m PGK in all physicochemical and enzymatic properties. After introduction of an initiation Met codon, and subcloning in an expression vector,

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STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

235

the modified T. maritima tpi gene (pTIM) supports the growth of a TIMdeficient E. coli strain on minimum medium. Furthermore, thermostable TIM activity can be detected in the crude extract (Adler, 1994; A. Hofmann, unpublished results 1995). Cloning of the complete fus gene is found to support the independent expression of PGK and PGK-TIM fusion protein in E. coli, indicating that the frameshift observed in T. maritima may be of general importance. In considering the amino acid sequences of PGK and the PGK-TIM fusion protein, reference is made to the nucleotide sequences of the pgk gene and the previously mentioned modified tpi gene with the Nterminal MITRK sequence. Because the corresponding engineered pTIM can be expressed in an active form in E. coli, it seems appropriate to use the amino acid sequence deduced from the modified tpi gene for sequence statistics and alignments. Figure 16 shows the sequence alignment of T m PGK with a number of homologous enzymes from mesophilic and thermophilic organisms. The computer-aided alignment was corrected by hand in order to replace surfaceexposed loops in the yeast enzyme by gaps. Most important in this context, in T m PGK, as in all other prokaryotic PGK sequences, the 15-residue “nose region” (residues 126-140) is lacking. Taking all known PGK sequences into account, 42 residues, or about 10% of an average PGK, are absolutely conserved.As identified by X-ray crystallography (Bankset al., 1979;Watson et al., 1982) and NMRstudies (Fairbrother et al., 1990), these comprise all residues involved in substrate binding and catalysis. The variable regions are spread over the entire sequence. However, taking the tertiary structure into account, it becomes clear that only solvent-accessibleresidues are variable, whereas the core region is highly conserved (Mori et al., 1986). Among the constant parts are the triple-glycine motifs (residues 234-236,369-371, and 392-394) and the 210-212 GGA sequence, both of which are believed to take part in the hinge-bending mechanism involved in catalysis. 3. Sequence Comparisons

In trying to correlate the amino acid exchanges to thermal stability, the predictions from previous statistical analyses (Argos et al., 1979) are even more ambiguous than in the case of GAPDH. The sequence identities range from 35% for Methanothermusfmidus (meye), a thermophilic archaeon (Fabry et al., 1990) to 61% for B. steurothamophilus (Davies et al., 1991); comparison with eukaryotic homologs yields about 50% identity. Attempts to correlate the amino acid composition to differences in thermal stability contradict the traffic rules to the extent that even the Lys + Arg exchange does not hold (Table VIII). In contrast to the

10 20 10 40 50 60 ---MEKEITIRDMLKGKRVIMRMFNVPVK--DGV-VQDDTAK-VILL thema ---WKKTIR.V.VRGK..FC.V.F.V.ME--Q.A-ITDM'.IRAALP.IRYLIEHGAK-VILA ---WKKTLK.I.VKGK..FC.V.F.V.MK--D.K-VTDET.IRAAIP.IQYLVEQCAK-VILA ------RTLL.L.PKGK..LV.V.Y.V.VQ--D.K-VQDET.ILESLP.LRHLLAGGA-SLVLL ---MSVIKMT.L.LAGK..FI.A.L.V.VK--D.K-VTSDA.IRASLP.IEW\LKQCAK-VMVT -SLSSKLSVQ.L.LKDK..FI.V.F.V.LD---.KKITSNQ.IVMLP.IKWLEHHPRYWLA --MFKFYTMD.F.YSGS&V.I.S.VDPHT.R-ILD~ENAKVAUA PA al

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bacst bacme theth ecoli yeast metfe SCC

thema bacst bacme theth ecoli yeast metfe 5ec

110 140 150 160 110 180 PGET--------------KNDPELAKFWASLADIHDAFOTAHRAHASNVGIA-QFIP-SVAG t h e m ffi.E--------------KNDPELAKAFAELADLY...GA...AHA.TE.IA-HYLP-AV.. bacs t ec.E--------------KNoPELAKAFAEW...GA...AHA.TE.IA-QHIP-AV.. bacme PC.E--------------KNDPELSARYARffiEAFL...GS...AHA.W.VA-RLLP-AY.. theth KG KDDETLSKKYMLCDVFVM...GT . . .AQA.TH.IG-KFADVAC.. ecoli IE .ffiSRKVDGOKVKASKEDVQKFRHELSSLAD~IN...GT...AHS.MV.FD---LPQRA..yeast metfe SE VLKRDPKVPBEIPtlLYBI(LSSVVDmN . . . M . .SQP.LV.FPLK-LP-S& . sec alV PE PF

120 2PO 210 220 210 240 FLMEKEIKFLSKVTYNPEKPYVWLGGAKVSDKIGVITNLMEK--ADRILIGGAMMFTFLKALF.MEK.LEVLGKALSNPDRPFTAII..A.VKDKIGVIDNLLEK--VLIIG.GLAYT.VKALF.MEK.LDVLSKALSNPERPFTAIV..A.VKDKIGVIDHLLDK--VLIIG.GLSYT.IKALF.MEK.VRALSRLLKDPERPYAWL..A.VSDKIGVIESLLPR--IDRLLIG.~FT.LKALP.LAA.LDAU;KALKEPARPMVAIV..S.VSTKLTVLDSLSKI--ADQLIVG.GIANT.IMQF.LEK.LKYFGKALENPTRPFLAIL..A.VADKIQLIDNLLDK--MSIIIG.GMAFT.KKVLE R.MER.vKTLyKIIKNvEKP. .V.I D D S I M ~ G S A D Y I PG PH aVll aVI aV

thema bacst bacme theth ecoli yeast ~ L ~ metfe sec

260 220 280 220 3Q0 25.0 G K E W ; S S - - - - R V E E D K I D ~ K E L L E K A K E K C V E I ~ P V D A V I A Q K I E P G V E K K ~ I D M I PtEh e m GHDVGKS----LLEEDKIELKSFMEKAKEKGVRFm.V.VWADRFANDANTKWPI-DAIPA bacst G H E V G K S - - - - L L E E D K I E ~ K S F M E K A K ~ V N F ~ . V . V W A D D F S N D A N I Q W S I E D - I Pbacme S GGEVGRS----LVEEDRLDLAKDLffiRAEALGVRVYL.E.WMERIEAGVETRVFP-ARAIPV theth GHDVGKS----LYEADLVDEAKRLL-----TTCNIPV.S.VRVATEFSETAPATLKSVND-V ecol i NTEIGDS----IFDKAGAEIVPKLMEKAKAKCVEWL.V.FIIADAFSADANTKT\PTDKEGIPA yeast ~ E K N R K I L Y R ~ G E 1 ( I L T - - - - N G K U R Y P I D D - I P N metfe sec aVlll alx PI PJ PK

310 320 310 3 40 35.0 360 3 20 GWnGLDIGPETIELFKQKLSDAKTVVWNGPMGVFEIDDFAEGTKQVALAIMLTEKGAITWGG DWSAL.I.PKTRELYRDVIRESKL VVW... M....MDA.IU(..KAIAEALAEALD--TYSVIG. DWEGL.A.PKTREIYADVIKNSKLVIW. . . M....LDA.AN..KAVAEALAEATD--TYSVIG. PmGL.I.PKTREAFARALEGARTVFW... M....VPP.DE..LAVGQAIAALEG--AFTWG. DEQIL.I.DASAQELAEILKNAKT1LW.. .V....FPN.RK..EIVANAIADSEA---FSIAG. CWQGL.N.PESRKLFMTVAKAKT1 V W . . . P....FEK.M..KALLDEWKSSMGNTVIIG. -FPIY.I.-REAKT-.. .A....EQQ.SI.-EDLLNAIASSN---AFSVIA. aX PL aXI 3 80 320 4PO 410 GDSMAVNKFGLEDKFSHVSTGGGASLEFLEGKELPGIASMRIKKK------ thema .DSAAAVEKFGLADKMDHI.T....SLEFHEGKQ..GWALEDK-------- bacst .DSAAAVEKFNLADKMSHI.T....SLEFMEGKE..GWALNDK-------- bacme .DSVMVNRLGLKERFGHV.T....SLEFLEKGT..GLEVLEG---------theth .DTLAAIDLFGIADKISYI.T....FLEFVEGKV..AVAMLEERAKK----- ecoli .DTATVAKKYGVTDKISHV.T....SLELLEGKE..GVAFLSEKK-------yeast . ~ G I S N K I M . S . , . . C T A F ~ E . A M Y L E E A R K R S D K Y I metfe axil PN aXlll aXlV SIX

thema bacs t bacme theth ecol i yeast metfe

sec

~

237

STRUCTURE AND STABILITY OF HWERSTABLE PROTEINS

TABLE VIII Amino Acid Composition of Phosphoglycerate Kinuses from Thennotoga maritima and /?om Other 0qani.sm.f Amino acid composition (mol %) Amino acid

thema

bacst

bacme

theth

ecoli

yeast

metfe

metbr

Ala cys Asp Glu Phe GIY His Ile

9.3

12.4 0.3 7.9 8.4 3.8 8.9 2.0 5.3 8.1 9.6 2.3 3.3 3.8

11.4 0.3 7.9 8.6 4.1 8.9 1.8 5.8 9.1 8.4 2.3 4.3 3.3 1.3 2.8 4.3 3.3 9.9 0.5 1.a 394

11.8 0.0 4.6 9.5 4.1 11.3 1.5 2.3 3.9 13.9 1.0 1.3 6.9 0.8 8.5 3.9 2.8 9.8 0.3 1.8 389

12.4 0.8 7.5 6.7 3.9 8.3 1.0 5.7 8.0 10.3 1.6 3.4 3.6 1.6 3.4 5.4 5.2 9.6 1.3 1.6 387

10.4 0.2 6.3 7.0 4.6 8.9 1.9 5.5 10.1 9.9 0.7 3.4 4.1 1.9 3.1 6.3 4.3 9.2 0.5 1.7 415

8.1 0.7 6.6 6.8 3.9 6.1 2.0 10.0 9.5 7.8 2.9 5.1 3.7 1.2 5.1 6.6 3.4 6.8 0.0 3.7 409

7.8 0.7 7.1 6.1 3.9 8.3 2.0 9.5 7.8 8.6 1.7 5.4 3.9 2.4 3.4 5.6 5.1 7.1 0.2 3.2 410

LYS

Leu Met Asn Pro Gln

4

Ser Thr Val TrP Tyr Total

0.0

5.5 9.3 4.3 9.5 1.5 6.5 10.8 9.0 2.5 2.5 4.5 1.5 3.3 4.3 3.8 10.5 0.8 0.8

399

0.5

5.1 2.8 3.3 9.6 0.5 2.0 394

a For abbreviations and references, see Fig. 16. metbr, Methanobactetium bryantii (Fabry et al., 1990).

PGK enzymes from Therrnus therrnophilus (Bowen et al., 1988),M. f m i d u s (Fabry et al., 1990),B. stearothaophilus (Davies et al., 1991),and Plasm& dium falciparum (Hicks et al., 1991), where thermal stability seems to correlate with the Arg content, Tm PGK has one of the lowest Arg contents, whereas the Lys content is one of the highest; considering the

FIG.16. Sequence alignment of phosphoglycerate kinases. thema, T. murifima(Schurig et al., 1995a);bacst, B. sfearothermuPhilus (Davies et al., 1991); bacme, B. megatnium (Schlapfer and Zuber, 1992);theth, T h u s thennophilus (Bowen et al., 1988);ecoli, Eschmchia coli (Alefounder et al., 1989);yeast, Sacchammyces mmisiae (Hitzeman el al., 1982);metfe, Methanothennusferuidus and metbr, Methanobacterium /nyantii (Fabry et al., 1990). Dots (.) represent identical amino acids, and dashes (-) denote gaps to align the sequences; OL and fi are secondary structural elements (sec), with the corresponding amino acids underlined; numbering of residues refers to the yeast structure (Watson et al., 1982).

238

R. JAENICKE ETAL.

sequence alignment, the Lys + Arg and Arg + Lys exchanges compensate one another. Similarly, the rules proposed by Menhdez-Arias arid Argos (1989) do not hold: The Ala content of the T. maritima enzyme is below average, with six additional Ala residues and four reversed exchanges in helical positions. Also, chemically labile amino acids such as Asn and Gln are not drastically reduced. The fact that there is n o Cys present in the enzyme cannot be considered as an essential adaptive mechanism faced with the anomalously high Cys content observed, for example, for TmGAPDH and LDH. It is obvious that the thermal stability cannot be explained without a high-resolution three-dimensional structure of the enzyme. Crystallographic analyses of Tm PCK and PGK-TIM are in progress. Regarding the TIM part of the sequence of the fus gene, the conclusions are similar to those drawn for the PGK part. Again, neither the amino acid composition nor the sequence provide a simple explanation for the thermal stability of the Tm enzyme (Schurig, 1995). Sequence identities between different TIM sequences range from 28% for the enzyme from the psychrophile Mmaxehand TTpanosomato 100% for human and chimpanzee. Figure 17 illustrates the corresponding sequence alignment for 5 of 30 known primary structures. It shows three active-site regions within all structures that are highly conserved (see Fig. 18): (i) residues 93-101, which include the anomalous His-97 with a pJ&below 4.0 (H95 in Fig. 18) believed to mediate the proton transfer to and from the triose phosphate hydroxyls; (ii) the flexible loop 6 (residues 165-180), which seals off the active site from the solvent during catalysis; and (iii) the a-helix dipole within the sequence 232-240 (loop 8, Fig. 18),which is involved in substrate binding (Lodi and Knowles, 1991; Sampson and Knowles, 1992). Whether the high frequency of the folding motif and the relationships among the various TIM barrels may be taken as stringent evidence for divergent evolution remains unknown. The strongest argument in support of this, namely, the absence of catalytic activity found for the TIM barrel protein narbonin, may not be conclusive, as the seed storage protein might have lost its original catalytic function, similar to taxonspecific enzyme crystallins in the eye lens (Branden. 1993; Eder and Kirschner, 1992; Farber, 1993;Jaenicke, 1994b). 4. Catalysis and Stability

The catalytic properties of PGK and the PGK-TIM fusion protein have been investigated in detail. The corresponding data are included in Table VII. To determine the rates of the catalytic reactions, TmGAPDH was used as an auxiliary enzyme in coupled assays for TIM (in the

239

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

El...... 10 1 1 1 1 1

al................ 20

RZ..... 30

a2........... 40

50

60

- -HRKPI IAGNWKMHKTLAEAVQFVEDVKGHVPPADEVISWCAPFLFLDRLVQ-TD

tpi-ixcs t ---ARTFFVOGNFKLNGSKQSIKEIVERI.NTASIPENVEWICPPATYLDYSVSLVKKPQ tpi_yeast -MSKPQPIAAhNVJKCNGSQQSLSELIDLFNSTSINHDVQCWASTNHLAMTKERLSHPK tpi-trybb M--RHPLVMGNWKLNGSRELVSNLRKELAGVAGCAVAIAPPEMYIDMAKREAEGSH tpi-ecoli MITRKLILAGNWKMHKTISEAKKFVSLLVNELHDVKEFEIWCPPFTALSEVGEILSGRN tpi-ttm

B3..

59 58 60 59 61

...

CONSENSUS

GA TGE S 130

119 118 119 119 121

G G H S E R R E

as....................

g5......

120

CONSENSUS

K

&6....

150

140

160

170

180

R G L I P I I C C G E S L E E R E S Q ~ K A ~ G L T P E Q V K Stpi-bacst S QGVGVILCIGETLEEKKAGKTLDWERQLNAVLEEW--WTNVWAYEPVWAIGTGLAA tpijeast SGFMVIACIGETLQERESGRTAVVVLTQIAAIAKKLKKACWAKVVIAYEPVWAIGTGKVA tpi-trybb QGLTPVLCIGETEAENEAGKTEEVCARQIDAVLKTQGAAAFEGAVIAYEPWVAIGTGKSA tpi-ecoli KGMTPILCVGETLEEREKGLTFCVVEKQVREGFYGLDKEEAKRWIAYEPWVAIGTGRVA tpi-ttm G

CGE

E

G T

a6...................... 190

200

V

Q

CONSENSUS B7... 210

al....*.. 220

58.a.

230

a8.e

240

TPEDANSVCGHIRSWSRLFGPEAA~IRIQYGGSVKPDNIRDFLAQQQIlX3PL~L tpi-bacst TPEDAQDIHASIRKFLASKLGDKAASELRILYGGSANGSNAVTFKDKADVDGFLVGGASL tpijeast TPQQAQEAHALIRSWVSSKICMVRGELRILYGGSVNGKNARTLYQQRDFLVGGASL tpi-trybb TPAQAQAV-HKFIRDHIAKVDI\NIAEQVIIQYGGSVNASNAAELFAQPDIM;ALVGGASL tpi-ecoli PQQAQEVH-AFIRKLLSEMYDEETAGSIRILYGGSIKPDNFLGLIVQRD1M;OLVOGASL tpi-ttm

........... 250

239 236 239 23 8 241

110

LK1CAQnmFADQGAYn;EVSPVMLKDU;VTWILGHSERRQMFAETDETVNKT tpi-bacst VTVGAQNAYLKASGAFTGENSMQIKDVGAKWVILGHSERRSYFHEDDKFIADKTKFALG tpi_yeast FVIAAQNA-IAKSGAFTGEVSLPILKDFGVNWIVLGHSERRAYYGETNEIVADKVAAAVA tpi-trybb IMLGAQNVDLNLSGAFTGETSARMLKDICAQYIIIGHSERRTYHKESDELIAKKFAVLKE tpi-ecoli IKLGAQNWYEDQGAFTGEISPLMLQEIGVEWIVGHSERRRIFKEDDEFINRKVKAVLE tpi-ttm

AQ

179 176 179 179 181

14...a4.......,............ 90 100

a3......... 80

70

EPASFLQLVEAGRHE--KPEFVDIINSRN-----KPEFVDIIKATQ-----KADAFAVIVKMEAAKQA KQSFIELARIHRGVIS--

I YGGS

N

CONSENSUS

260 tpi-bacst tpijeast tpi-trybb tpi-ecoli tpi-ttm

CONSENSUS

FIG. 17. Sequence alignment of triose-phosphate isomerase. The a helices (a)and p strands ( p ) are marked above the sequences. The three conserved consensus regions are underlined. Numbers in the text refer to T. maritima numbering. bacst, B. stearothermophilus; yeast, Saccharomyces cerevisiae; trybb, Trypanosom bruce' bruck; ecoli, Eschen'chia coli; tun, Thennotoga maritima. For a complete sequence alignment and the corresponding references of all known TIM amino acid sequences, see Wierenga d al. (1992).

240

R JAENICKE ETAL.

flexible loop 6

active site pocket

loop 5

loop 4

phosphate binding helix

loop 3

loop 2

FIG.18. The triose-phosphate isomerase barrel fold. H1 to H8 and B1 to B8 refer to a helices and B strands within the units. Black dots mark positions of interface residues in the Ttyponosumu structure. The long arrow points to the catalytic residues K13,H95, and El67 in the center of the barrel. Modified after Wierenga el al. (1992).

glycolytic direction) and for PGK (in the gluconeogenic direction). The instability of dihydroxyacetone phosphate and NADH renders the determination of the optimum and maximum temperatures of the catalytic reactions for both enzymes difficult. However, extrapolating the half-times of thermal inactivation to higher temperatures, and considering the intrinsic stability of other T. mantima enzymes, the temperature limit of thermal denaturation for the two enzymes is likely to exceed the optimum growth temperature of the bacterium (see below). The pH optima, specific activities, and activation energies are in the same range observed for other PGK and TIM enzymes under the respective physiological conditions. One striking difference between the two PGK active enzymes is the higher catalytic efficiency of monomeric PGK compared with the fusion protein, measured in both directions, glycolysis and gluconeogenesis. Because domain movements have been suggested as an essential element of the catalytic mechanism, the reduced activity of the PGK-TIM fusion protein would lend support to the notion that this domain movement is hindered in the bifunctional enzyme complex. The difference is also reflected in the k;. values for the substrate Sphosphoglycerate (3-PG),

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

241

which confirms the observation that under physiological conditions enzymes do not necessarily operate with maximum efficiency, probably because the multiple functions of proteins (folding, targeting, catalysis, degradation, etc.) can hardly be optimized simultaneously. In this connection, it is remarkable that the two catalytic sites of the fusion protein differ in their temperature dependences, with activation energies for TIM and PGK differing by a factor of 2 (Fig. 19A; Table WI). As in other hyperstable enzymes investigated so far, PGK and the PGK-TIM fusion protein require high temperatures to exhibit significant catalytic activity. In the mesophilic temperature regime, both enzymes are essentially inactive, in accordance with the concept of corresponding states. In the case of TIM, the high activation energy of the catalytic reaction (80 kJ/mol) leads to an extrapolated value of approximately 2 X lo4 U/mg at the optimum growth temperature. This high catalytic efficiency is in agreement with the activity reported for the enzyme from mesophilic sources at the respective growth temperatures (Noltmann, 1972). The normal catalytic activity supports the idea that in the fusion protein TIM is present at least as a dimer. From the three-dimensional structure it is obvious that two subunits are required for the correct positioning of the catalytic residues Lys-13 and His-95; mono-TIM shows only 0.1% of the activity of the natural TIM dimer (Borchert et al., 1994). To quantify the thermal stability of PGK and the PGK-TIM fusion protein, irreversible heat inactivation was monitored in the temperature range between 70 and 105°C (Fig. 19B). Both enzymes undergo thermal denaturation at lOO"C, measured as loss of PGK activity. The lower transition temperature of TIM (88°C) seems to reflect specific buffer effects. From the point of view of the maximum growth temperature of T. marilima, both enzymes are clearly sufficiently stable to carry out their function under in uivo conditions, without requiring extrinsic stabilizing factors.

D. Enolase

1. General Properties Enolase (EC 4.2.1.11, phosphopyruvate hydratase; 2-phospho-~-glycerate hydrolase) catalyzes the dehydration of 2-phospho-~-glycerate(2-PG), yielding phosphoenolpyruvate (PEP). The ubiquitous metalloenzyme is one of the most abundant proteins in yeast and other organisms (Wold, 1971; Brewer, 1981;van der Straeten et al., 1991).Yeast enolase has been studied in detail regarding its catalytic mechanism. The dimeric enzyme

242

R. JAENICKE ETAL

16

24

32

40 48 56 64 Temperature ("C)

12

B

Temperature ('0 FIG. 19. Thermal stability and catalysis of T. manfima phosphoglycerate kinase and phosphoglycerate kinase-hose-phosphate isomerase fusion protein. ( A ) Temperature dependence of the PGK ( 0 ) and TIM ( 0 ) activity of the Tm PGK-TIM firsion protein. Inset: Arrhenius plot. (B) Thermal stability of Tm PGK ( 0 ) and 7'm PGK-TIM fusion protein [both PGK ( 0 ) and TIM activity ( A ) ] . Activity measurements wrrr done under standard conditions.

needs 2 mol of tightly bound divalent cations, a conformational one and a catalytic one. The physiological cofactor is Mg'+, but other ions such as Mn2+,Ca", Zn'+,Ni2+,andTb2+also bind, some of them even with higher a n i t y . The conformational metal ion is required for substrate

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

243

binding. Its removal causes spectral changes and alterations in subunit interactions, apart from a decrease in thermostability (Hanlon and Westhead, 1969). Only Mg2+and Zn2+promote catalysis, with the turnover rate determined by the second, catalytic, metal ion. If a nonactivating ion (e.g., Ca2+)binds to the conformational binding site, no catalysis is detectable. Regarding the state of association, most enolases have been shown to be 90-kDa homodimers. This holds for the eukaryotic enzyme (from yeast to human), as well as the enzymes from E. coli and Pyrococcus furiosus (the latter being the only archaeal enolase investigated so far) (Wold, 1971; Peak et al., 1994). In contrast, some homooctameric enolases have been isolated from the bacterial domain, including a number of thermophiles (Stellwagen et ad., 1973; Barnes and Stellwagen, 1973). Crystallographic studies on yeast apoenolase showed that the enzyme belongs to the family of ap-barrel enzymes like triose-phosphate isomerase (Stec and Lebioda, 1990). However, in contrast to the (ap)sTIM barrel, the second p strand in the enolase barrel is antiparallel to the other strands, and the first a helix is antiparallel to the other a helices, resulting in an ~rapp(a/3)~ topology. As in other apbarrel enzymes, the active sites lie in cavities at the carboxylic end of the inner p barrel. X-Ray analysis of the ternary enolase/2-PG/Mg2+complex and the corresponding enolase/phosphoglycolate/Mg2+ enzyme-inhibitor complex confirm the sequential catalytic mechanism with a trigonal bipyramidal coordination of the oxoligand around the conformational metal ion as an essential element for catalysis (Lebioda et al., 1991). Fluoride is known to be a potent enolase inhibitor. Apart from blocking the glycolytic pathway, it inhibits also the highly specific PEPdependent phosphotransferase system (Hata et al., 1990). An explanation of the synergistic effects of fluoride and phosphate ions was provided by the crystal structure of the quaternary enolase/Mg2+/F-/P0,- complex (Lebioda et al., 1993). 2. Enolase jkom Thenotoga maritima Enolase is one of the most abundant proteins in T . maritima and can be easily purified to homogeneity (Schurig et al., 1995b). As shown by atomic absorption, the enzyme contains firmly bound Mg2+that can be removed by dialysis against EDTA. The N-terminal sequence shows a high degree of similarity to eukaryotic enolases and to the enzyme from Pyrococcusfuriosus (Schurig et al., 1995b). Currently no sequence data of thermostable enolases are available; hence, an analysis of the intrinsic stability of the T. maritima enzyme on the basis of the alignment comparison of sequences is not feasible. Comparison of the amino acid compositions of enolases from (hyper-)thermophiles and mesophiles with respect

244

R JAENICKE ETAL.

to hydrophobicity, arginine content, etc., does not provide conclusive results (Barnes and Stellwagen, 1973; Peak et al., 1994). Enolase from T. man'tim shows high intrinsic stability, with thermal transitions at 90 and 94°C in the absence and presence of Mg2+,respectively (Fig. 20A).As usual, the high thermal stability is paralleled by an anomalously high stability against chaotropic denaturants: taking the residual activity or fluorescence emission as a measure, the transition

B

A

60

80 90 100 Temperature ("C)

70

2Wr

"0

20

40 60 80 Temperature ("C)

100

20 30 40 50 6 0 7 0 Tempcntun (TI

FIG.20. Thermal stability and caralysis of T. man'h'ma enolase. (A) Thermal stability after 2 hr of incubation at the given temperatures in the absence ( 0 ) and presence (0) of 5 mM MgCI, (50 mM HEPES buffer, pH 7.5). (B)Temperature dependence of the specific activity for the 2-PG dehydration reaction, measured in TrisHCl buffer, pH 7.5, at M e concentrations adapted according to the temperature dependence of &,MW2+: 13-25"C, 10 mM MgCI,; 26-45OC, 5 mM MgCl,; 50-65"C, 1 m M MgCI,; 70-95"C, 0.5 mM MgC12. Inset: Arrhenius plot. (C) Effect of temperature on K,n;rppfor 2 P G ( 0 ) and Mgl+ ( 0 ) .

STRUCTURE AND STABIIJTY OF HYPERSTABLE PROTEINS

245

midpoints of the guanidinedependent denaturation are shifted from about 0.5 M in the case of the rabbit muscle enzyme to 3.0 M for the enzyme from T. maritima. Comparison of the two irreversible unfolding transitions reveals that deactivation precedes denaturation. As shown by chromatographic and hydrodynamic methods, the enzyme belongs to the family of homooctameric enolases with a molecular mass of the active enzyme of 345 2 10 kDa and subunits of 48 2 5 kDa. Ultracentrifugal analysis of the native apoenzyme yields a sedimentation constant of 13.4 S; addition of Mg2+as cofactor leads to a 5% decrease, indicating minor rearrangements and/or changes in hydration on ion = 1.403) clearly points to an binding. The frictional coefficient (fh oblate shape of the octamer, with an axial ratio of around 9. Denaturation at pH 2.0 and in the presence of 4 M guanidinium chloride leads to a compact “A-state” intermediate (7 S), on one hand, and the fully denatured polypeptide chain (2 S), on the other (Schurig, 1995). Insight into the topology of the native quaternary structure was obtained from image processing of negatively stained electron micrographs. In accordance with the sedimentation analysis, the enzyme is an oblate ring 17 nm in diameter with 4fold symmetry, equivalent to a tetramer of dimers (Fig. 21).

FIG.21. Octameric quaternary structure of T. matitima enolase. (A) Field view of the negatively stained enzyme (3%uranyl acetate (w/v)). Bar: 100 nm. (B) Average over 799 aligned particleswith contour lines superimposed. (C) Orientational correlation function of (B). For experimental details, see Schurig et al. (1995b).

246

R. JAENICKE ETAL

With the effect of subunit association on protein stability (Jaenicke, 1991a) and the dimeric quaternary structure of rnesophilic enolases (Wold, 1971) in mind, it is tempting to ascribe the high intrinsic stability of the T. maritimenzyme to its octameric quaternary structure. However, two findings clearly contradict this hypothesis: (i) there are thermally unstable octameric enolases in mesophiles (Singh and Setlow, 1978; Kaufmann and Bartholmes, 1992),and (ii) enolase from the hyperthermophilic archaeon Pyrococcus furiosus has been reported to be a dirner (Peak et al., 1994). Thus, there is no clear correlation of the stability of Tm enolase to its state of association.

3. Catalytic Properties By monitoring the transformation of 2-PG to PEP at 230 or 240 nm at varying concentrations of divalent ions (Schurig et al., l995b), it was found that Mg2+is essential and optimally effective for catalytic activity. The pH optimum (pH 7.0-8.0) and temperature dependence of the reaction do not deviate from enolases from mesophilic sources (Wold, 1971; Stellwagen et al., 1973; Schurig et al., 1995b). Specific effects of buffer anions are attributable to Mg2+complex formation. They may be quenched by the addition of excess Mg4+to the assay mixture. This does not hold for the phosphate and fluoride inhibition, where a more complex mechanism prevails (Brewer, 1981). As has been mentioned, these two ions exhibit synergistic effects: in the absence of phosphate, fluoride shows noncompetitive inhibition, whereas in the presence of phosphate the inhibition becomes competitive (Schurig et aL, 1995b). The K,,, values for 2-PG and Mg2+show a pronounced temperature dependence: raising the temperature from 13 to 75°C leads to a decrease in K,,, by a factor of 2 and 30, respectively (Fig. 20C). Beyond 45°C the K,,, values remain practically constant, and at physiological temperature both reach plateau values close to the level observed for the enzyme from mesophiles at room temperature. Again, the previously mentioned corresponding states situation holds. As has been shown for other T. mritimaenzymes (e.g., GAPDH and LDH), the leveling of K,,, to an upper plateau value at elevated temperature cannot be generalized (Hecht et al., 1989; Wrba et al., 1990b). Enolase from T. man'tima has an optimum temperature for catalytic activity at about 90°C. which exceeds the optimum growth temperature of the organism by about 10°C (Fig. 20B). The Arrhenius plot is nonlinear, indicating some change in conformation or in the mechanism of the enzyme at around 45"C, with limiting activation energies of 75 and 43 kJ/mol, respectively. In this connection, it might not be fortuitious that the K,,, value becomes independent of temperature above 45°C

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

247

(see above). At optimal temperature, the specific activity of the enzyme reaches approximately 2000 U/mg; corresponding values for the mesophilic homologs are -70 U/mg for the enzyme from B. megaterz’um, 160 U/mg for E. coli, and 450-900 U/mg for T h u s aquaticus (Schurig et al., 1995b). Obviously, during evolution, Tm enolase succeeded in optimizing more than one function, exhibiting both maximum catalytic efficiency and stability at the upper limit of the physiological temperature regime.

-

E. Lactate Dehydrogenase 1. Homologous Lactate Dehydrogenase Lactate dehydrogenase (LDH, EC 1.1.1.27) is one of the most extensively studied enzymes in terms of structure-function relationship, folding, stability, reaction mechanism, and evolution (Holbrook et al., 1975; Rossmann et al., 1975; Jaenicke, 1987). It has been the paradigm of isoenzymes as permutations of different gene products in one and the same quaternary structure, which is commonly tetrameric with a subunit molecular mass of about 35 kDa. The enzyme catalyzes the last step in anaerobic glycolysis, namely, the conversion of pyruvate to lactate with concomitant oxidation of NADH; stereochemically the enzyme may be or D specific (EC 1.1.1.27 or 1.1.1.28). Prokaryotic L-LDHenzymes are homotetramers with dissociation constants in the nanomolar range. In a number of organisms they depend on Mn2+and/or fructose 1,Gbisphosphate (FBP) as allosteric effectors that regulate carbohydrate metabolism (Garvie,1980).Great efforts have been made to understand the structural basis of temperature adaptation at the protein and DNA level, by comparing LDH enzymes from psychrophiles, mesophiles, and thermophiles (Argos et al., 1979; Zuber, 1981, 1988). So far no conclusive results have been obtained, in spite of the fact that high-resolution crystal structures of mesophilic and thermophilic homologs are available (Table IX) . To unravel the mechanism of thermal stabilization,sequence comparison and protein engineering have been applied. In the first approach, attempts were made to define thermal stability in terms of increments attributable to single amino acid or nucleotide exchanges (Zuber, 1981, 1988; H. Zuber, personal communication 1994). In brief, thermophilic LDH enzymes exhibit increased hydrophobicity and ion pairing, which can be correlated with a specific pattern of base substitutions at the DNA level. The mutational approach has been based mainly on the high-resolution structure of LDH from B. stearothenophilus. Here, conser-

248

R JAENICKE ETAL.

TABLEIX Available h h ' n Data Bank Film of lockat ate Lkhydmpases (M a1 mid 1995)' PDB

R (A) code

Source

Ligands

B. stcomthmnophilus (T)

Apoenzyme NAD/FBP NADH/oxamate/FBP FBP/ Cop NADH

2.8 3.0 2.5 3.0 2.0

NAD/pyruvate Apoenzyme

SLDH White pf 01. (1974) 6LDH Abad-Zapatero et al. ( 1987) 2.1 lLDM Griflith tf al. (1987) 2.8 8LDH Abad-Zapatero ef al. ( 1988) 2.96 2LDX Griffith tf al. (1987)

LoclobaciUw cask (M) Bijidobaclmum hprn (M) Squalw acanthias (P)

+

NAD/oxamate Apoenzyme/citrate Mw mwculus (M) Sw s m j a (M)

Pig heart Pig muscle

Thmnus cahphilus (T) Themwtogta mantima (HT)

Apoenzyme NAD/ (S)-lactate NADH NADH/oxamate Apoenzyme NADH/oxamate/FBP

lLDG SLDB lLDN 1LLC 1LLD

Accession reference Piontek ef al. (1989) Piontek Pt al. (1989) Wigley ef al. (1991) Buehner ef al. (1988) lwata rf al. (1992)

3.0 2.0

2.7 2.2 2.0 -2.5 -2.8

SLDH Grau d al. (1980) SLDB Dunn ef a/. (1991) 9LDT Dunn ef al. (1991) - No entryb No entry'

-

R, Resolution of X-ray analysis; PDB, Protein Data Bank. Physiological temperature ranges: HT, hyperthermophilic; M, mesophilic; P, poikilothermic; T, therrnophilic. bPreliminary crystallographic data ( b i d e et oL, 1991). Preliminary crystallographic data (Ostendorp el ~ l .1996). , I

vation of the topology of the threedimensional structure in different species allowsvariationsof stabilitytoward higher and lower denaturation temperatures to be correlated with site-directed mutations. To test the assumption that thermal stability can be enhanced by reducing the hydrophobic surface area, peripheral hydrophobic residues were changed into neutral or polar ones, and the rate of thermal denaturation was reduced significantly (Wigley et al., 1987; Kotik and Zuber, 1992, 1993). To avoid side effects on the catalytic efficiency (often observed in single point mutations), other experiments in the same direction made use of hybridization, for example, grafting the N-terminal domain of Bst LDH with the Gterminal domain of LDH from the mesophilic B. megutenurn. Again an increase in stability was obtained, this time without changes in enzymatic properties (Waldvogel et al., 1987). Attempts to ascribe specific functional or stabilizing properties to certain stretches of the polypeptide chain do not seem to provide un-

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

249

equivocal results, as single amino acid exchanges and their reverse mutations do not compensate one another (Zalli et al., 1991, 1992). An example that may explain the inconsistencies in the case of Bst LDH are the changes caused by the double mutation Glul02Arg-Argl71Tyr, where binding of stabilizing sulfate ions and enhanced hydrophobicity increase the denaturation temperature by 20°C (Kallwass et al., 1992). Numerous similar studies, including rational protein design and subsequent exploration of insertions and amino acid exchanges, have been reported (Dunn et al., 1991;Jackson et al., 1992). The wide diversity of effects illustrate the unlimited structural and functional potential of proteins without unveiling the molecular basis of their stability. 2. Thennotoga Lactate Dehydrogenase: Physical and Enzymatic Properties Initially Tm LDH was purified from cells grown in large-scale fermentor (Wrba et al., 1990b). Because of the low level of expression under standard anaerobic growth conditions (MMSmedium, 80°C), however, the gene had to be cloned and expressed under a strong promoter in E. coli in order to obtain the enzyme in amounts sufficient for physical characterization (Ostendorp, 1996). Cloning was accomplished by complementation of an E. coli double mutant lacking pyruvate formate-lyase and DLDH activities. Expression of the gene in E. coli yielded a protein that was indistinguishable from native T m LDH (Ostendorp et aL,1993). Its physicochemical properties are summarized in Table X,which also includes relevant catalytic data. The Michaelis constants for the substrate and NADH increase drastically with temperature, reaching millimolar concentrations under physiological conditions (Hecht et al., 1989). The increase is less pronounced in the moderately thermophilic and mesophilic LDH enzymes from B. stearothaophilus and pig heart, and it occurs even in the presence of the allosteric activator FBP, which is known to improve K,, by charge repulsion along the subunit interface (Cameron et al., 1994; Iwata et al., 1994). As the FBP binding sites are located between the subunits, and thermal stabilization is strongly dependent on subunit interactions, the influence of FBP on the activity and thermal stability of the hyperthermophilic T m LDH is not surprising. The enzyme has the same tetrameric quaternary structure as most known LDH enzymes (Long and Kaplan, 1968). Molecular mass determinations of the native tetramer and its subunit yield values of 144 and 36 kDa, respectively. The molecular weight of the polypeptide chain calculated from the amino acid sequence is 34,994. Ultracentrifugal analysis confirms the tetrameric state of association for the temperature range between 4 and 30°C and for concentrations as low as 0.5 p M . The catalytic activity shows a broad maximum between pH 4.0 and 8.0. At

250

R. JAENICKE ETAL.

TABLE X Properties of Honwlogous Mesophilic and Thmnophilic Lactate Dehydmpases

Property Molecular mass (11.4) ( m a ) Subunit mass (MI) ( m a ) Thermal denaturation (holo, "C) Deactivation in GdmCl (c,,? 20°C) Denaturation in GdmCl ( c l t p20°C) Specific activity (U/mg) ("C) pH optimum of catalysis K (~Mpyruvate)("C)'

Y (PMNADH)

( O W '

Optimum reactivation at O°C/?300C

T. mritim 140 35

91 1.6 M 2.1 M 3700 (80) pH 6.0 16 (20) 20 (35) 60 (55) 850 (80) 1.5 (20) 2 (35) 27 (55) 575 (80) 20%/85%

B. steamthffmophilus

Pig heart

140 35 60 1.0 M 1.5 M 2200 (55) pH 6.0 98 (20) 150 (35) 280 (55)

140 35 45 0.5 M 0.5 M 400 (20)

50 ( 2 0 ) 50 (35) 50 (55)

10 (10) 10 (35)

-

-

pH 6.0

60 (20) 107 (35)

-

-

35%/85%

a K, under optimum conditions of the assay at temperatures (in brackets): T. mantama, 0.1 M imidazole, pH 6.0, 3 m M FBP, 5 m M EDTA; B. steumthermophalus, 0.1 M TEA. pH 6.0, 0.3 m M FBP, 5 m M EDTA; pig heart: 0.1 M phosphate, pH 6.0, 1 m M EDTA,

1 m M dithioerythritol.

the pH optimum (pH 6.0),long-term incubation of the a p e and holoenzyme (in the presence of excess NADt) yields midpoints of thermal denaturation at 70 and 91"C, respectively. Addition of FBP leads to a 3fold increase of the half-time of thermal deactivation at 90°C; NADtand FBP act synergistically, with an 8O-fold increase in ( t1,2)wo(:. This means that the enzyme is sufficiently stable to be fully active in its natural environment. Extrinsic factors may provide additional stability. Short-term incubation in the standard assay allows the catalytic activity to be measured up to 100°C (Fig. 22). The temperature dependence of the rate shows Arrhenius behavior with an activation energy of 114 kJ/mol. No significant curvature is detectable (Ostendorp et al., 1996).Judging from the drastically increased K,,, values for the substrate and NAD', the optimization of catalytic function and thermal stability has its limits. Similar observations for other extremophilic enzymes confirm that under physiological conditions proteins do not necessarily operate with maximum catalytic efficiency.

3. Sequence Alignment and Homology Mo&ling To minimize general species differences and to explore possible rules of thermal stabilization in the case of the hyperthermophilic enzyme,

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

= ,E

8000

f

2

I

x ->

c

8 u

4.-

5

6000 LOO0

2000

251

?I

a,

a

c " o

0

20

LO 60 Temperature

80 [OC

1

100

FIG.22. Temperature dependence of the specific activity of mesophilic and therrnophilic lactate dehydrogenases.Standard assays were done under optimum conditions with ( A ) pig heart LDH, (0) Esl LDH, and ( 0 ) Tm LDH (Hecht et al., 1989; Ostendorp d al., 1996).

1 1 prokaryotic LDH enzymes with different physiological temperature optima were selected and compared with respect to composition and sequence (Fig. 23). As in the other T. mantima enzymes investigated so far, there is no preference for specific nucleotides at the level of the gene; the GC content for all triplets is 48.6%, and that for the third codon, 52.8%, in contrast to the ldh gene of Thennus caldophilus where the GC content of the third base was reported to be higher than 95% (Kunai et al., 1986). As has been mentioned in the case of GAPDH, comparison of the amino acid composition does not reveal obvious shifts between mesophilic, thermophilic, and hyperthermophilic species. Among the preferred exchanges in going from mesophiles to thermophiles (Argos et al., 1979), the only correct prediction is the preference for arginine, which is increased in all thermophilic homologs. That it cannot be generalized is obvious from the composition of Tm PGK, where the arginine content is lowest among all homologs in the database (Section V,C) . Given that Tm LDH has the maximum number of cysteine residues (5 per monomer) among all prokaryotic LDH enzymes, the argument that thermophiles avoid Cys because of its susceptibility to oxidation can also be ignored. The idea that the Cys cluster (C213, C216, C219) as part of the flexible surface loop (213-220) might exert an effect on the assembly and stability of the tetrameric quaternary structure (in terms of the formation of cystine cross-links) can hardly be relevant considering the reducing cytosolic environment and the observation that under oxidizing conditions the enzyme loses its activity. With 319 amino acids, the polypeptide chain of Tm LDH is somewhat longer than that of T h m u s LDH but compares well with all other sequences (Table XI). Pairwise alignment yields a high similarity of over

A

-

5

6

1

9

0

10

11

A

8.8

14.2

14.8

10.1

11.4

10.5

10.3

9.1

9.2

8.2

14.0

10.9

C

1.6

0

0

0.6

0.6

0

0.3

0.6

0.9

0.9

0.3

0.9

D

5.3

4.5

3.5

6.6

6.6

8.0

9.1

6.3

5.1

5.7

6.1

5.9

E

1.2

1.1

7.7

6.0

6.9

5.2

4.1

6.0

6.6

7.5

6.4

5.9

F

3.4

2.6

2.3

4.4

4.1

3.4

3.1

3.8

3.8

4.1

5.2

2.2

G

8.1

9.4

10.0

8.8

9.1

6.8

9.4

8.2

9.2

8.5

7.9

7.8

R

2.2

1.9

1.9

2.5

2.2

1.8

0.6

3.1

2.8

1.6

1.e

2.5

I

7.8

4.2

3.2

8.8

8.2

9.2

6.9

9.1

1.6

9.1

8.2

8.1

I

6.6

2.3

1.6

4.4

4.1

7.8

7.2

5.3

1.3

5.7

6.4

5.0

L

9.1

11.6

11.9

7.3

7.3

7.8

a.4

6.9

7.9

8.5

7.9

7.8

n

2.2

0.6

1.0

1.9

2.2

2.8

1.9

2.2

0.9

2.8

N

3.8

1.0

0.6

5.0

3.8

5.0

6.0

6.6

4.9

5.0

P

3.1

4.2

5.2

2.8

3.8

3.1

3.2

3.1

3.0

5.3

0

2.2

3.5

2.6

2.2

3.1

4.1

2.5

2.5

4.3

3.4

R

5.6

8.4

8.4

5.1

3.4

4.1

1.9

4.1

2.1

3.8

S

5.6

4.2

5.2

4.1

3.4

6.2

1.2

4.4

4.1

4.1

4.6

5.3

T

4.1

4.5

3.9

3.8

4.1

5.5

4.4

6.0

5.1

6.0

4.0

6.6

V

9.4

12.3

12.6

9.5

8.8

8.0

0.4

7.5

9.2

6.3

1.9

8.4

w

0.3

1.0

0.6

0.9

1.3

0.9

0.3

0.6

0.6

0.9

0.6

0.6

r -

2.8

2.6

2.9

3.8

3.8

3.1

3.4

3.1

3.8

3.8

2.1

1.6

M

319

310

310

317

317

325

310

318

316

318

328

32a

FIG.23. Amino acid compositions and sequence alignment of prokaryotic lactate dehydrogenases. (A) Amino acid compositions (mol%). (B) Sequence alignment, with numbering of the enzymes as in (A): Ttm, T. &ti% 1. T h w aguutim; 2 , T k muc cokiqbhiluc; 3, 8. steamthmnophiluc; 4, B. caldotenax; 5 , Lactobarillus cask; 252

B

40

30

50

60

83

70

90

Ttm M - - - - - - - - - K I G I M ; f f i R ~ S ~ A F ~ G F A R P N L I S 1 M--- - - ----FGIV. S .F . SATAYALVLQXAR .vvLMLDR--FLA@HAE. ILIWTP.A-2 M----- ----WG?X. S .M. SATAYALRLLGVAR WLVDLDR--FLA@HAE. 1LWTP.A-HPWWWGSY3 MK"---GGARVWI.A.F. .ASYVTALlNSIAD. I V L I D A N E - - S K A I o M M . E M E K v . A P K P V D ~ 4 MKK---RI;M(VAW.T.F. ..VYAFALlNSIAD.IVLI~--NXAEDM.~.APKPAD~ 5 PISIT-D~~LV.D.A..SSYAF~IAQ.IGIVDIFK--Q(TKGDAT.LShlALP.TS-PKKIYSACISDAKDA 6 M S S N - P - N X Q ~ V .D.A . .SSYAFMWXSIAE. FvFJDVW--DRTIGW. -.TA-PKKIYSGEf7 MKWTPKTRRVAVI . T . F. . SSYAFSMVNPGIAN.LVLID--PXAEliEAR. INFX3W.AT-M8 MN-K---HVNKVALI .A. F . SSYAFALIWITD.LvvIDUJK--EKAK.pvM. L W i G K A . ~ P V X T S Y ~ 9 MKQR-- - " A L I . A. S . . SSYAFALLNSITE. LVIIDLNE--NKAMjMM. LNWXV.-SDCWA 10 mAT--KQHKKvILV. D.A . S ~ A L ~ I A Q . ~ I I E I ~ ~ ~ W . L S H A L A . T S - P K K T Y ~ 11 MW3-TWWITLAVI .A. A . .STLAPAAAPRGIAR. IVLEDIAK--ER~.E9K;SS.YPNSIDCSDDPEICRDA

. .

.

. .

PA 100 105 T b 1 2 3 4 5 6 7 8 9

10 11

120

110

pc

oc

PB

132

150

140

AB

160

110

W V T V A A G V P Q K P I A R N V S W A P D S M ~ ~ P S K V F G ~ A R R W N A . VAQR. .ET. Q. L D R " v p K I L K A A p u \ v L L I A T . ~ A y R L s o L p P E U W . I. .TA. R A W . .VAQR. .ET. Q. LDRNRPVFAQVVPRW!?AVLLVAT VMMvAyRLSALsFGRW. . .I. TA. I L T Y A W SGLpHEI(vI.... I . . T A . D L W I C . . W K . ET. .D. VDRNIAIFRSIVESWSTQXnVAT D L W I C . .hNQK. .ET. D . V I B ( N ? . A I F R S F C G L F L V A T . ILTYAWSSLFQ. I . TA. ILTYAlWl&3FPKNRVV. S. TA. D L W I T . .APXQ. .ET . .D.VEP(NLKILKSIVDPIVDSIFLVAA. DLWIT ..AFQK..ES D.VM("ILS~~VKPWDSGFDOI~VAA....ILTYA'RIKPS....S..SS. DLAVIT. .ANOA. ET. .D .VEl@MCIF€CIVKDIMNSIILVAT I L A H V I Q ~ . .I. .TA. D N C I C , .hNQK..ET. .E .VEKNIXIFKGIVSEWASGFEIF%IAT ILTYAWT . SA. D N C I C . .ANQI(..ET. D . V E K N L R I F K G m P I F L I A T . ILTYAIwI(psYjLF'KERI1. . .I. .lG. D L W I T .AFQK. .ET. D.VGKNLAIM(SIVKQVIESIFLVAA. I L T Y A W S W P W IATiNAQKLTG~IF P N Y I T . .PRQK. .QS . .E .V G A T V N I L Y M L I T .

.

. .

.

. . . ..

...

. .

.. . . .. .. .

-

OLVE

act.rite 180

. .. . ... ... .. ....

.. . .. .

-OD

Ttm 1 2 3 4 5 6

aa

190

PE

200

.

209 210

.

-PF

UlF 224

ABCDAB

. . .

230

240

L R T L I A Q H C G F S ~ G ~ S ~ ~ I ~ I P ~ - I M K C - D S ( I ~ ~ ~ I I P R K G A T F .ALLRQHLLvApQS . H A W . S . V L W S S . P V G C M W F A Q - A R G R A L T P ~ . R . Ec.. F .ALLRp((LRvApQS .HAYVL. S.VLWSS.QVGGVPLLEFAE-ARGF@LSP-. . R . . Ec.. F.FU&FXFsvApoN.HAYII.. . T . L P V W S Q . Y I G V M P I R - S K G E - ~ .Q. ~ . .Fx.

.. .. .. ..

. . .. . .. . .. .. . F.FLIGDYFAVAPlN.HAYII.....T.LPVWSP.DIGGVPIR-SKGE-~WNVPD&..Q..M.... F.QSIAP3VMIOARS.HAYIM.. . . . T . F P V W S H . N I G G V P I - m - I K E D X L - . .E. .KL. . . . L.VALGKQFNWPRS. DAYIM. , . ..S. F A A Y S T . T I G R + P V R D - V A - K B X - V ~.D. . .NL. . . . I F .YLLSllYFEVDsRN.HAY IN.. . ..T .FFJWSH .QIGGVKLEWIN-TAhI-EKEP-. .H. .NR. .. . 8 F.FMSEWGAAPW. MI1 . . . . . T . LPVWSH.NVGGVWSELVE-KNM-YKQEELCQ~..T. .M. .. . 9 F.FLLGFIFDIAPAN.HAYII.....T.LPVWSH.DIGGISITIK-PNPE-~INVPM..Q..EK.... 10 .-.F HAYIM. . . . .S .FAWSH.--OVPN-RI-. .T. .NU.. . . 11 L . n v m r m m m . m ~ m . .. . .S.VPLWES.TI~IPLEGKD-IKQ~. .K. . m . . . . - -PG

LlZF 250

260

prr

pH

210

280 285

UlG12G

flfX.100p

290

301

310

320

330

AB

Ttm H Y A I A L A V A D I V E S I F F D ~ V L ~ W I ~ ~ S A S 1 I J " E I T ~ S 1 Y .GIGRGLARLTRAILTD~GVFTVSLFTPEW.VEZVALSL. RILGRR.VEATLYPRWEE. PQhtiUl. E I .wLAsALoF 2 Y .G I G A G L A R L V R A I L T D E K ~ P E V E.VLEVSLSL. R I W .vEoI1pIpSLSpE.REWSkI. E I EAAF-F 3 Y . G L A K j L R R ~ L k " k I L T V S A Y L K L Y.ERDwIGV.AVIMIN. 1 P . N I E I E L M I D . m . .AT. WURAETR 4 Y . G L A K j L A R - L I L ~ ~ Y L K ~ . ~ I G V . A V 1P.NIEIUDEE.KKWEHR. ~. .AT. .FvuRyFK) 5 F.GIATALARISRAIIPL~Y.INDLYIGT.AVIMIN.IQNILEIPLTDH.EESM)K..~..KVLTOAFAI(MIETRQ

. . . .. .

6 7 8 9

10 11

F.GIGTALMRISRAILRPV3x~Y.LM)IYIGT.AII'XT.LKQIIFSPLSAD.~D..AT..~ Y .GLAKjLVRITKAILEUl~SIL?1ISALLEGQY. ISWYIGV. AIINKN.vRpIIELNLTPH . a m ..S I . .WTUiRAW Y . G V A M S L A R I T K A I L H N p I S I L ~ .ADwYIGV.AvvNRo. Y I A G I M . K E Q F L J I . .GV..NIIXPHFVN F . G I A K ; L A R I T K A I L N N E N S V L W S l Y L E G E Y . ~ . A W & S N1REIVeLTLNep.RWFIM. . .NV..EIF . G I A V A L A R I T K A I L I L P ~ ~ Y . F N E V F .KE. I . .AIIDEaFwAAA?&W N . A I Q 4 S G V D I I E I L F V S S M L K D ~ .ISDICMSV.TLLNRQ.VNM'I~?~'FVSIX.LAALJVL..ET..ETAAQPSF a3G

PK

-BL

PM

w

FIG. 23. (Continued) 6 , Lactobacillus plantarum; 7, B. megatm'um; 8, B. subtilir; 9, B. psychmsarrharolyticus; 10, Streptococcus mutans; 1 1, Bijidobach'um lagum. For references, see Ostendorp et al. (1993). Dots (.) denote identical amino acids; dashes (-) represent deletions; a and /3 are secondary structural elements referring to &t LDH (Piontek el al., 1990). Residues numbered according to Eventoff et al. (1977). 253

254

R. JAENICKE ETAL.

SO%, suggesting that the overall topology of the enzyme is closely related to the backbone structure of homologs whose threedimensional structure has already been solved at high resolution. The expected deviations in the core of the enzyme are predicted to be less than 0.7 (Hilbert t t al., 1993). Taking all available data into account, an extended alignment exposes highly conserved positions within the respective sequences of eukaryotic and prokaryotic LDH enzymes. In four of these positions ( Ile-24, Lys-43,Ser-91,Va1-138),TmLDH adopts the eukaryotic sequence. However, the highest values are obtained comparing the 7. man'lima enzyme with the one from T h u s uquaticus (Top,-85"C, 48% identity, 62% similarity) and B. psychrosaccharolyticus (Top,-25% 44% identity, 61% similarity). Another candidate for this kind of comparison that is presently under investigation is the enzyme from the Antarctic fish Dissostochus mansoni ( Tphyslol -1.9"C, T,, -5°C) ( C . Marshall, personal communication 1995). Whether the principles leading to structural

A

TABLE XI

PainVire Alignment of Amino Arid Sequence of Thmnotoga maribma !.-lartatuIIPhydrogenase (319 Residua) with Known LDH Enrvmef'

Organism

Number of amino acids

Identical amino acids

Identity (%)

Similarity (%)

7hennus aquaticus B. psychmsaccharolytiws BiJidobactm'um longurn* B. caldotmax Lactobacillus cak* Thennus cald@hilus B. subtilis B. mgalenum B. s&arothmn@hilus* Lactobacillus pkantarum Streptococcus mutam

310 318 320 317 325 310 316 318 317 320 328

153 139 129 135 134 149 135 132 126 130 136

47.96 43.57 40.31 42.32 41.23 46.71 42.32 41.38 39.50 40.63 41.46

61.97 61.09 60.91 60.56 60.44 60.31 60.31 60.07 59.98 58.08 58.59

............................................................... Mouse testis* Dogfish (apoenzyme)* Dogfish*

33 1 323 329

120 115 121

36.25 35.60 36.78

53.20 53.12 52.13

Prokaryotic sequences appear above dotted line; asterisks (*) refer t o LDH enzymes

with three-dimensional srructures solved at high resolution. Data calculated according to Lesk el al. (1986), based on the PAM 250 matrix proposed by Dayhoff

pt

al. (1983).

STRUCTURE AND STABILITY OF HYPERSTABLE PROTEINS

255

adaptation to high and low temperatures have anything in common remains to be shown. As mentioned, homology modeling yields a threedimensional structure closely similar to the one known from previous determinations (Auerbach, 1994). Preliminary high-resolution X-ray data seem to confirm the modeling approach, again demonstrating that temperature adaptation represents the cumulative effects of small changes within the margin of average structural fluctuations (G. Auerbach, personal communication 1996). On the basis of the conserved overall topology of the enzyme, analysis of the amino acid residues involved in binding the substrate and coenzyme shows that almost all residues contributing to binding sites are conserved. An exception is the charged amino acid residue Arg-31 in the T. mantima enzyme. Compared to the mainly hydrophobic residues in the homologous sequences, again, only the cold-adapted B. psychrosaccharolytics also contains a hydrophilic side chain (Ser) in this position. Increased hydrophobicity in the core of the molecule, especially in the subunit interfaces, may be concluded from the local accumulation of Phe residues in helices a l F and a3G (see Fig. 23B). Contributions to the intrinsic stability have also been attributed to additional proline residues in loop regions, where the stabilizingeffect depends on the (entropydriven) destabilization of the denatured state (Suzuki, 1989). In the case of T m LDH all prolines are found to occur in mesophiles and thermophiles. They probably do not play a significant role in the stabilization of the native state. 4. Structure and Structure F m a t i o n

As has been mentioned, sequence alignment and molecular modeling suggest the overall topology of T m LDH to be close to the experimentally determined threedimensional structure of the enzyme from B. stearothermophilus. In extending this work, an attempt was made to predict the structure in more detail by homology modeling based on the highresolution structures of the enzymes from B. stearothmnophilus, Bijide bacterium longum, and Squalus acanthias, and the programs SYBYL 6.1, INSIGHT 11, and PROCHECK (Auerbach, 1994). An energy minimization of the model was carried out with 100 steps steepest descent and 1000 cycles conjugate gradient with a cutoff radius of 16.0 A. As a result, the secondary structure content is estimated to 46% a helix and 18.8% /3 sheet, in agreement with experimental data from circular dichroism spectra. Compared with other LDH enzymes, this value is slightly increased. The C-terminal a helix seems to be extended and fits into a groove in the surface of the molecule. The volume of the complete tetramer amounts to 152,040 As and reveals in total 117 cavities with a

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Connolly radius of 1.4 A. This value is close to the one observed for the enzymes from pig and dogfish. It also confirms the crystallographicresult for Tm GAPDH (KorndBrfer el aZ., 1995) (Section V,B), suggesting that the increased rigidity of the thermostable enzyme cannot be ascribed to the reduction of the number of cavities. Instead, the stabilization must be based on minute local improvements of van der Waals contacts within the whole molecule. For the inner core of the molecule, this corresponds to the enthalpic contributions to hydrophobic interactions (Privalov, 1990), whereas in the periphery ion pairing seems to play a significant role. As illustrated in Fig. 24, the predicted structure does not contain any residues in disallowed regions of the Ramachandran diagram; more than 85% occur in the most favored regions. This value is close to the characteristics of good quality models: On the basis of analysis of 118 structures of at least 2.0 A resolution and Rfactors no greater than 20%, these would be expected to have about 90% residues in the most favored regions. On the other hand, files of the known high-resolution X-ray structures in the Protein Data Bank show that there are torsion angles outside the allowed regions (Table MI). Thus, it may be assumed that the predicted structure is close to the real one. However, it is clear that a high-resolution crystal structure is required in order to confirm the model and to explain exchanged residues in terms of possible strategies of thermal stabilization; such work is in progress. With respect to structure formation, two points are of interest, first, expression of the hyperthermophilic active protein in E. colz and, second, the mechanism of in vitro folding and association. The expression and purification of recombinant Tm LDH was performed aerobically and at room temperature. Obviously, these conditions are unphysiological to the extreme, since in vivotranslation,folding, and assembly occur anaerobically at about 80°C. The purification procedure makes use of the extreme thermal stability of the recombinant enzyme, removing the host proteins by heat denaturation. Nevertheless, the recombinant protein is expressed as active authentic enzyme with all its physicochemical and enzymatic properties indistinguishable from the natural Tm LDH. The conclusion to be drawn from these observations is that translation and selfarganization of the thermophilic protein do not require the natural high-temperature conditions: Folding and assembly lead to the native protein at a temperature almost 60°C below the optimum growth temperature of T. muritimu (cf. the analogous observation for GAPDH in Section V,B). In connection with the mechanism of folding and association, kinetic analysis of the in vitm reconstitution after preceding denaturation may

257

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Phi (degrees) FIG. 24. Ramachandran diagram of the T. man'tima lactate dehydrogenase model. Glycine residues are represented by triangles. Residues 192, 197, 199, and 200 were adopted from the B. stearolhophilus structure.

PROChXCK Analysis

of

TABLE XI1 Ramachandran Plots of Lactate Dehydrogaasef

PDB code

Organism

1 LLD 9LDT 1 LDM 1 LDN 1 LLC

Bifidobacterium longum Sus srrofa Squalus acanthias B. stenrothennophihs Lactobacillus cask 7'. maritima

-

Resolution

% residues in regions that are

(4

Central

Allowed

Disallowed

2.0 2.0 2.1 2.5 3.0 Model

91.1 88.7 88.7 84.3 57.9 85.7

8.9 10.8 10.9 15.8 38.6 14.3

0.0 0.5 0.3 0.0 3.5 0.0

Analysis was done with known high-resolution structures and the model calculated for Tm LDH. Glycine and proline, as well as the Cterminal 10 amino acid residues of the model. are not considered.

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be assumed to mimic relevant steps of enzyme self-organization in the cell (Jaenicke, 1993). Reconstitution of mesophilic LDH enzymes has been shown to follow a sequential uni-bimolecular kinetic mechanism with dimers as inactive intermediates. The ratedetermining steps are formation of the structured monomer in the course of a first-order folding event and second-order association of the preformed dimers (Jaenicke, 1987). In the case of the moderately thermophilic B. stearothermophilus LDH, a different mechanism holds. Here, the reactivation kinetics are independent of protein concentration, indicating that a unimolecular isomerization reaction must be the rate-limiting step. Neither the coenzyme, NADH, nor the allosteric effector FBP affect the kinetics (Miiller et al., 1984).The LDH from T. man'tima shows the same behavior. Chemical cross-linking experiments allow the slow folding reaction and the diffusioncontrolled association to be identified (Ostendorp, 1995). As a result, the self-organization of the oligomeric hyperthermophilic enzyme seems to be determined by slow folding reactions; the rate of assembly of the monomeric and dimeric intermediates, even at temperatures below the physiological level, is close to diffusion controlled.

VI. CONCLUSIONS The viability of T. maritima and other hyperthermophilic microorganisms at temperatures close to the boiling point of water is based on the inherent thermostability of their cell inventory. In many cases, this is assisted by extrinsic components such as coenzymes, metabolites, or specific effectors. For a number of hyperthermophilic archaea (e.g., Fyrodictium, Sulfolobus),chaperones have been proposed to serve as additional safeguards; in the case of T. mantima, however, accessory proteins are expressed at low levels only (G. Frey, personal communication 1996). Rapid turnover of intrinsically unstable components by de novo synthesis does not seem to play a significant role in accomplishing thermophily. The molecular basis of macromolecular thermostability is still unresolved. It seems to reside in small variations of weak interactions that overall give rise to altered packing at the level of domains and subunits. In extreme cases, one single point mutation or the exchange of one single atom in a critical nucleic acid base were found to be sufficient to cause drastic shifts in the denaturation temperature (Brock, 1986). The overall enhancement of stability for a given protein then results values from numerous small mutational changes accumulating to AA Grmb of the order of only a few kilocalories per mole. As a consequence, no clear-cut strategy of thermal adaptation has been extracted from the vast amount of sequence data that have been accumulated. Going one

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step further, there is no reason to expect general rules that might be applied to quantitatively predict protein stabilizationfrom specific amino acid exchanges. However, thanks to incisive research of a whole generation of physical biochemists, insight into the interactions involved in protein stabilization has grown to the extent that substitutions at specific sites of a known structure can be correlated with positive or negative The breakthrough came from the observation that changes in AGSmb. sitedirected mutagenesis allows amino acids to be exchanged without necessarily altering the overall structure of a given protein. In this context, systematic studies referred especially to hydrophobic substitutions, charge changes, and helix dipoles (Alber, 1989a; Nicholson et al., 1991; Matthews, 1993). From the examples discussed in this article, it is obvious that each protein shows individual characteristics in terms of its activation properties, flexibility, temperature dependence of K, and k,,,, etc. Specific activities or K,,, values of homologous enzymes from mesophiles and thermophiles in many cases have been found to be closely similar under the respective physiological conditions. Evidently, in the course of evolution, temperature-sensitive mutations adjusted both stability and biological activity. Whether the relationship between protein stability and protein function generally follows the above-mentioned model of T4 lysozyme (Shoichet et al., 1995) needs further investigation. In all cases reported for T. mantima enzymes, the proteins were found to be intrinsically stable up to temperatures beyond the optimum growth temperature of the bacterium; extrinsic factors, if they show significant effects, merely serve as a reserve. Covalent modifications that have been reported as an additional mechanism of protein stabilization (Oshima, 1979) seem to be absent in T. muritima. As indicated by the position of hyperthermophiles in the three kingdoms of life, the old evolutionary problem of who came first has apparently been solved: all the very early branches off the phylogenetic tree represent hyperthermophiles (Woese, 1993). From this, the assumption seems reasonable that thermophiles were primordial and that subsequent organisms were derived from them, extending the biosphere from limited terrestrial and marine volcanic areas into less extreme mesophilic zones and, finally, over the whole globe. The limiting factors that determine the maximum growth temperature are clearly defined by the stability of macromolecular cell components and metabolites. On the other hand, the observation that cell division of hyperthermophiles vanishes at temperatures around 50°C needs still to be explored. Apparently, the kinetics of catabolic and anabolic processes become dislocated as a consequence of differences in the respective activation energies. Also,

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at lower temperatures, the functionality of membranes may be limiting as a consequence of altered fluidity, permeability, and protein or lipid interactions. Considering the average temperature of the oceans (-4”C), cells may survive in a dormant state, even in the presence of oxygen. This way, hyperthermophiles may spread, transported not only by currents in the sea but also in the cold atmosphere. Life at high temperatures has been the subject of biochemical research for almost 150 years. It was mere curiosity that initiated Hoppe-Seyler’s first experiments (Perutz, 1995).In the meantime, problems of microbial evolution, ecology, and industrial application, apart from fundamental interest in protein folding and protein stability, changed the field into a gold rush scenario. However, basic questions remain unanswered. What is the upper limit of viability for pro- and eukaryotes, what determines macromolecular stability, is there a genetic control of thermal adaptation, are there specific structural properties of thermophilic membrane proteins, how does high temperature modlfy the folding pathway of proteins, what is the mechanism of chaperone action, how is the thermal stabilityof molecular assemblies promoted, what are the adaptive mechanisms of membrane fluidity, and how do protein-lipid interactions influence thermoadaptation? It is obvious that most of these problems also await a solution for Eschen’chia coli and the rest of the mesophilic world. Nevertheless, attacking a problem in a comparative fashion has often turned out to be the only viable approach, and covering a wide range of values for potential variables may magnify minute effects so that they become detectable as significant quantities. For this reason, work reported in this article has to be continued in order to create a database that should allow general conclusions to be made with respect to one or the other of the above fundamental questions.

ACKNOWLEDGMENTS Work performed in the authors’ laboratorieswas supported by Grants of the Deutsche Forschungsgerneinschaft, the Fogarty International Center for Advanced Studies (National Institutes of Health, Bethesda, MD), and the European Community. We thank ProfessorK. 0.Stetter for continuoussupport in all questions connected with microbiologi d techniques and fermentation. Fruitful discussions with Drs. R. L. Baldwin. S. Daopin, M. E. Goldberg, K Kirschner, C. N. Pace, P. L. Privalov, R. Seckler, and G . N. Somero are gratefully acknowledged.

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