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Protein engineering strategies in examining protein folding intermediates
Protein engineering strategies in examining protein folding intermediates Jill A. Zitzewitz and C. Robert Matthews The Pennsylvania State University, University Park, USA Mutagenesis has proven to be a powerful tool for examining the structures and stabilities of intermediates which appear during the folding of globular proteins. Combining site-directed mutagenesis with other, more classical, methods of protein chemistry, such as chemical labeling and cleavage, has advanced the investigation of these transient, marginally stable species. The high cooperativity of the folding reaction has been reduced by mutagenesis to produce a series of reactions involving stable, highly populated, partially folded forms. New insights into inherently slow processes, such as proline isomerization reactions and disulfide bond rearrangements, have also resulted from protein engineering experiments. Current Opinion in Structural Biology 1993, 3:594-600
Introduction Site-directed mutagenesis has become a routine method for analyzing the influence of individual amino acid side chains on the structure, function, and stability of proteins [1o]. The information obtained from these studies has been used to re-engineer existing proteins to improve stability or catalytic activity [2°]. Yet the design of entirely new proteins with novel functions remains a major challenge in protein engineering. A serious difficulty in ab initio design is the inability to predict successfully the unique, functional three-dimensional conformation of a protein from its amino acid sequence. Fortunately, protein engineering strategies have also provided new tools to aid in deciphering the rules governing protein folding. The two major impediments to solving the protein folding problem are the high cooperativity and rapidity of the folding react/on. The highly cooperative nature of the folding reaction results in very low equilibrium concentrations of partially folded forms which could provide mechanistic dues. In cases where intermediates are highly populated during the folding reaction, these species often exist for only a few milliseconds. The use of conventional spectroscopic methods to determine their structures and stabilities is simply not feasible. These two obstacles are now being overcome with the advent of several new technologies, including genetic engineering, peptide synthesis, two-dimensional NMR, and stoppedflow circular dichroism (CD). Most of these technological advances have been reviewed recently [3,4oo-6o.]. The purpose of this short review is to survey a number of papers that highlight current advances in protein engineezing strategies for examination of protein folding intermediates. Related issues of protein stability and of fold-
ing and functional modules are discussed in accompanying reviews in this section by Matthews (see pp 589-593) and Landro and Schimmel (see pp 549-554), respectively.
Protein engineering strategies for probing folding intermediates Over 30 years ago, Anfinsen and his colleagues [7] proposed that the linear sequence of amino acids determines the final three-dimensional structure of a protein. A corollary to this hypothesis is that the structure and relative populations of intermediates along the folding pathway, and thus the mechanism of protein folding itself, are also dictated by the primary protein sequence. Thus, changes in the amino acid sequence can potentially alter the structure and the stability of both the final conformation and any folding intermediates. The replacement of particular amino acids in a protein by site-directed mutagenesis offers a unique opportunity to determine the role of these amino acids in the folding process and to test a given protein folding hypothesis. Detailed discussions of such mutagenic analyses can be found in reviews by Matthews [8], Goldenberg [9] and Matouschek and Fersht [10]. Mutagenesis can also be used to incorporate conformational probes at specific sites for studying local folding events. For example, the fluorescence properties of genetically inserted tryptophan probes can be used to characterize protein folding intermediat6s in tryptophanfree proteins [11]. Alternatively, folding can be followed by 19F NMR after growth of tryptophan-containing proreins in the presence of 6-fluorotryptophan [12-]. Mutant proteins which contain unique chemically reac-
Abbreviations BPTI--bovine pancreatic trypsin inhibitor; CD---circular dichroism; PRAI--phosphoribosylanthranilate isomerase.
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Strategies in examining protein folding intermediates Zitzewitz and Matthews tive groups can also be modified to incorporate acceptor/donor pairs for distance measurements in intermediates by fluorescence energy transfer [13]. For example, a cysteine-specific fluorescent probe, such as 5-[ [2-[ (iodoacetyl)amino] ethyl] amino] napthalene 1-sulfonic acid (IAEDANS), can be used as an energy acceptor of donor fluorescence from a single tryptophan residue [14°]. A major challenge of such experiments is to incorporate the probe at a position which does not cause significant disruption of native structure. Cysteine residues, introduced by site-directed mutagenesis, can serve as chemically reactive probes for studying local folding events [15,16]. Probes are introduced into hydrophobic regions which are inaccessible to solvent in the native structure. The chemical accessibility of the cysteine sulfhydryl group to reaction with iodoacetate or 5,5'-dithiobis(2-nitrobenzoate) at different stages of folding can be used, in favorable circumstances, to give a thermodynamic and kinetic description of the folding mechanism in the vicinity of the probe. This method has recently been used to characterize protein folding intermediates in human carbonic anhydrase [17°°] and yeast phosphoglycerate kinase [18.o]. An important control in these experiments is the measurement of the rate of modification of the reactive group in the completely unfolded protein or, preferably, in a peptide containing the cysteine residue. Naturally, the concentration of the reagent and the inherent reactivity must be such that the labeling process occurs more rapidly than subsequent refolding or potential unfolding reactions. Protein fragments can be generated by mutagenesis, either by incorporating new start and stop codons into the structural gene or by introducing specific chemical cleavage sites into the amino acid sequence. Analysis of the equilibrium and kinetic properties for folding of these fragments can identify autonomous folding units and test their role in the folding process [19°]. Care must be taken with this approach in interpretation of the results because the conformations adopted by the fragment may not be the same as those found for the same sequence embedded in the fuR-length protein. For example, the interaction of two or more autonomous folding units may induce further structural changes in the protein. A dramatic example of this phenomenon is the cyclosporin A/cyclophilin system where the cyclosporin A peptide turns inside out upon binding to cyclophilin [20]. Other aspects of protein folding can be studied by using mutagenic methods to make changes in the primary structures of proteins, such as by the Insertion [21] or deletion [22] of loops, or by the construction of disulfide bonds [23°]. More drastic changes, including translocation of the amino and carboxyl termini [24,25], module reshuffling [26] and production of tandemty linked proteins [27°], can also provide new insights into the mechanism by which the sequence guides the folding reaction. Selected protein engineering strategies for studying distinct features of protein folding mechanisms are described in the following sections.
Early intermediates in folding ,. The evidence that has accumulated over the past few years suggests that unfolded proteins (whether unfolding was induced by heat, acid or denaturant) are not completely devoid of residual sttucmre [28]. Recently, Gottfried and Haas [29 "°] measured fluorescence energy transfer between an amino-terminal donor probe and an acceptor probe attached to one of the four lysines of bovine pancreatic trysin inhibitor (BPTI). Their results indicate that BPTI does not exist as a statistical random coil, even under strongly denaturing and reducing conditions. Non-local interactions, responsible for the compact structure in unfolded BPTI, are implicated as essential elements in directing the formation of early folding intermediates. The earliest observable intermediates in protein folding, detected by stopped-flow CD and pulse-labeling NMR, form in less than 5ms and contain substantial nonpolar surfaces and secondary structure [6"']. These very early intermediates are probably an ensemble of conformations in dynamic equilibrium and may contain nonnative elements of structure. Protein fragments may prove useful for kinetic characterization of early intermediates because fragments cannot form the native long-range interactions responsible for supersecondary and tertiary structure formation. In effect, the folding reaction is halted at the stage where all the information contained in the fragment has been processed. Goldberg and colleagues [30o] have recently used stopped-flow CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence spectroscopy to show that a carboxy-terminal fragment of the [3 subunit of tryptophan synthase folds in less than 4 ms into a condensed state with non-native secondary structure. The authors conclude that non-specific collapse of a comparable condensed state occurs with the whole protein before reaching the stage at which the first intermediates with native-like secondary structure are observed.
Intermediates that fold directly into the native conformation The rate-limiting step in the folding of many proteins involves the formation of the native conformation. Protein engineering strategies have provided a great deal of insight into the structure of the intermediates that lead directly to the native conformation and the intervening transition states. In many cases, access to this information is enhanced because the rate-limiting step in unfolding is the reverse of the rate-limiting step in refolding. Unfolding is usually a much simpler kinetic process than refolding, making comparison of the properties of the transition state with the native conformation relatively straightforward. The rate-limiting step in the denaturant-induced unfolding and refolding of the at subunit of tryptophan synthase (Fig. 1) involves the interconversion of the native conformation and a stable folding intermediate. This stable intermediate has significant secondary and tertiary structure in the amino folding unit (residues 1-188; 13-strands 1--6)
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Engineering and design but was originally thought to have a disordered carboxy folding unit (residues 189-268; [3-strands 7-8) [31,32]. More recendy, comparisons of single and double mutants at the interface between the folding units have demonstrated the existence of residual structure in the carboxy folding unit, particularly in the vicinity of residues 175 (strand 6) and 209 (strand 7) in the intermediate [33"]. Thus, the final phase in folding appears to involve the docking of strand 8 to strand 1 along with a global repacking of many buried side chains to form the native structure.
Fig. 1. Ribbon diagram of the (x subunit of tryptophan synthase. The eight ~strands that form the core of the barrel are numbered consecutively. The amino folding unit (light grey) comprises 6strands 1-6 and a-helices 0-5, and the carboxy folding unit (dark grey) comprises 13-strands 7-8 and (x-helices 6-8.
Breaking down the cooperativity of the folding reaction A natural consequence of the high cooperativity observed in the folding of globular proteins is the low population of partially folded forms. Mutagenesis provides a potential method for breaking down this cooperativity and for populating folding intermediates. Mutations at the interface between the two folding units of the 0t subunit of tryptophan synthase selectively destabilize the native conformation relative to the stable intermediate [37]. The effect is to enhance the population o f the intermediate in the unfolding transition zone. A similar observation has recently, been made in mutagenic studies invoMng the dimeric protein tryptophan aporepressor (CJ Mann, CA Royer, CR Matthews, unpublished data). Replacements of single tryptophan residues at the dimer interface result in a non-coincidence of the unfolding transitions detected by fluorescence and CD. It appears that these replacements selectively destabilize the interdimer association reaction without greatly perturbing the stability of a folded monomer. The results discussed above, and others [38,39], suggest that the high cooperativity observed for the folding of globular proteins may be a consequence of strong interactions between two or more autonomous folding units. By using mutagenesis to break down the cooperativiw of the folding reaction, folding intermediates can be populated and, thus, a more detailed understanding of the information contained in individual segments of a polypeptide can be obtained. Further insights into how this information is modulated when the segments are annealed in the native conformation can be obtained from additional mutagenic and fragment studies on the structures and stabilities of these partially folded forms.
Disulfide bond rearrangements Fersht and coworkers [34"] have conducted an elaborate mutagenic analysis of the rate-limiting step in the unfolding/refolding pathway of bamase. An extension of the reaction coordinate analysis approach was used to probe the structure of the rate-limiting transition state relative to the native and unfolded forms [35"]. The parameter ¢, introduced to provide a quantitative description of the transition state, equals zero when the transition state is native-like and equals unity when the transition state resembles the unfolded state. Because there is no simple linear relationship between ~ and the extent of structure formation, fractional values of ~ are more difficult to interpret. This type of analysis nevertheless provides useful information about the structure of the rate-limiting transition state for refolding, which, as noted above, is identical to that for unfolding in this instance. The consolidation of the main hydrophobic core, the closing of many loops, and the capping of the amino termini of the helices are the final events in the folding of bamase. Interestingly, a kinetically significant intermediate (or possibly ensemble of intermediates) on the refolding pathway of bamase has a much less stable core than the transition state that leads to the final conformation [36"].
The oxidative folding of reduced BPTI is one of the most extensively studied protein folding mechanisms [40--]. BPTI is a small, single-domain protein which is stabiLized by three disulfide bonds. The rate-limiting step in folding involves a disulfide bond rearrangement prior to the formation of the native structure. Efforts to elucidate the mechanism of folding for BPTI have involved the characterization of partially disulfide-bonded intermediates trapped by iodoacetate during the oxidative refolding of BPTI [41,42]. Protein engineering strategies have been used to synthesize analogs of these trapped intermediates for structural characterization. By replacing cysteines not involved in disulfide bonds with serines or alanines, analogs of the five intermediates retaining one or two of the native disultides were engineered [40 o-]. Four of these analogs, including the (5-55) single-disulfide intermediate [43,44], adopt native-like structures as assessed by NMR spectroscopy [45,46] or X-ray crystallography [47]. An analog of the other crucial single-disulfide intermediate, (30-51), has a native-like [3-sheet and at-helix [48..], as was suggested by earlier studies of a peptide model [49]. Yet the first 14 residues contain little ff any residual structure and residues 37-41 appear to be highly flexible [50"]. Thus,
Strategies in examining protein folding intermediates Zitzewitz and Matthews the (30-51) single-disulfide intermediate may be an example of a true folding intermediate because it contains regions of both native and unfolded structure.
Proline isomerization In 1975, Brandts and his coworkers [51] proposed that the c i s - t r a m isomerization of the peptide bond preceding proline is responsible for the slow refolding of proteins. In this model, only the population of unfolded protein ha~_ng the same isomeric configuration as the native protein is able to fold directly into the native structure. The population of molecules containing non-native proline configurations first has to isomerize its X-Pro (where X represents any amino acid) bonds before folding. Prolines in the c/s conformation in the native protein are often responsible for slow folding reactions (see below). The trans conformation around the peptide bond is expected to be preferred in the unfolded protein based on the predominance of t r a m proline isomers in unstructured peptides [51,52]. Brandts' proline hypothesis is supported by mutagenic studies on several proteins in which the key proline(s) are replaced by other amino acids which isomerize rapidly in the unfolded protein. Replacement of what are often c/s proline residues has led to the loss of a slow refolding phase in several proteins, including thioredoxin [53], ribonuclease T1 [54], ribonuclease A [55 °°] and staphylococcal nuclease [56°°]. Although more difficult to predict, proline isomerization reactions which lead to t r a m prolines in the native structure can also result in slow folding phases, as observed for yeast iso2-cytochrome c [57] and a mutant of BPTI containing only two disulfide bonds [58]. Also, the refolding kinetics of staphylococcal nuclease appears to exhibit phases attributable to both c/s and t r a m configurational isomerization [56"']. Interestingly, a proline-free mutant of human lysozyme exhibits the same denaturant-independent slow refolding kinetic phase observed for the wild-type protein, indicating that this reaction cannot be attributed to proline isomerization [59]. Thus, it is possible that the cis-trans isomerization of other peptide bonds besides those involving prolines may account for some of the slow isomerization reactions observed in protein folding. It should be noted that the significant majority of proline residues in a protein are not involved in rate-limiting isomerization reactions. Wolfenden and coworkers [60..] recently observed that the rate of the c i s - t r a m isomerization of acetyiproline N-methylamide is enhanced in the non-polar solvent toluene. The authors suggest that the isomerization of proline residues buried in the hydrophobic interior of a protein occurs more rapidly than folding reactions. An alternative mechanism for intramolecular catalysis of proline isomerization has been suggested by mutagenic studies of dihydrofolate reductase [61--]. An arginine side chain that is distant in the primary sequence from the critical proline residue, but adjacent to it in the three-dimensional structure, is responsible for this catalysis, presumably by forming
hydrogen bonds with the imide nitrogen, and/or the carbonyl oxygen of the proline. A survey of hydrogenbonding patterns in other proteins reveals a significant number of candidates for this type of acceleration of the proline isomerization reaction. Additional mutagenic studies are required to test the generality of this intramolecular catalysis mechanism.
Conclusion As illustrated by the examples above, protein engineering methodologies provide a very powerful tool for mapping the structure and stability of folding intermediates and their roles in protein folding reactions. Enhanced understanding of critical concepts in protein folding is promised by further applications of protein engineering strategies that combine the tools of recombinant DNA technology with more classical protein chemistry methods. The development of a folding code, analogous to the genetic code, may eventually allow the three-dimensional structure of a protein to be predicted from the linear amino acid sequence. This information would be invaluable for the de novo design of new proteins with novel functions. Furthermore, identification of those sequence features which are critical for successful progression along the folding reaction pathway may help avoid problems of aggregation or proteolysis, ultimately leading to improved yields of products for the biotechnology industry.
Acknowledgements Work from this laboratory is funded by the National Institutes of Health (GM23303) and the National Science Foundation (DMB9004707). JA Zitzewitz is supported by NIH Postdoctoral Fellowship Award GM14954. The authors also kindly thank Deborah Bebout for critical reading of this manuscript.
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vAN MIERLO CPM, DARBY NJ, CREIGHTON TE: The Partially Folded Conformation of the Cys30-Cys51 Intermediate in t h e Disulfide Folding Pathway of Bovine Pancreatic Trypsin Inhibitor. Proc Natl Acad Sci USA 1992, 89:6775-6779. An analog of the single disulfide-bonded intermediate (30-51) of BPTI, containing serine replacements for the other four cysteines, has a nativelike secondary structure in the core and a disordered amino terminus. Thus, the intermediate reflected by this analog may be the only well populated disulfide-bonded intermediate in the folding pathway of BPTI which is a true folding intermediate.
•o
49.
OAS TG, KIM PS: A Peptide Model of a Protein Folding Intermediate. Nature 1988, 336:42-48.
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vAN MIERLO CPM, DARBYNJ, KEELERJ, NEUHAUSD, CREIGHTON TE: Partially Folded Conformations o f t h e ( 3 0 - 5 1 ) Intermediate in t h e Disulphide Folding Pathway of Bovine Pancreatic Trypsin Inhibitor. tH and 15N Resonance Assignm e n t s and Determination of Backbone Dynamics from 15N Relaxation Measurements. J Mol Biol 1993, 229:1125-1146. Residues 1-14 and 37-41 are highly flexible in this (30-51) folding intermediate of BPTI. This flexibility may explain why any of the possible disulfide bonds involving Cys5, Cysl4 or Cys38 can be formed from this intermediate during folding.
•
51.
BRAND'rSJF, HALVORSONHR, BRENNANM: Consideration of the Possibility that the Slow Step in Protein Denaturation Reactions Is Due to Cis--Trans Isomerization of Proline Residues. Biochemistry 1975, 14:4953-4963.
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GRATHWOHLC, W{YrHmCH K: T h e X-pro Peptide Bond as an NMR Probe for Conformational Studies o f Flexible Linear Peptides. Biopolymers 1976, 15:2025-2041.
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KELLEYRF, RICHARDS FM: Replacement of Proline-76 with Alanine ElimInates t h e Slowest Kinetic Phase in Thioredoxin FoldIng. Biochemistry 1987, 26:6765-6774.
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KIEFHABERT, GRUNERTH-P, HAHN U, SCHMID FX: Replacement of a CIS Proline Simplifies t h e Mechanism of Ribonuclease TI Folding. Biochemistry 1990, 29:6475-6480.
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SCHULTZDA, SCHMID FX, BALDWINRL: Cis Proline Mutants of Ribonuclease A. II. Elimination of t h e Slow-Folding Forms by Mutation. Protein Sci 1992, 1:917-924. O n the basis of a careful kinetic analysis of the unfolding/refolding of single- and double-proline mutants, the authors conclude that prolines 93 and 114, the two cis prolines in the native conformation of ribonuclease A, are jointly, responsible for forming the major slow-folding intermediate
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Engineering and design NAKANOT, ANTONINO LC, FOX RO, FINK A L E ect of Proline Mutations o n t h e Stability a n d Kinetics of Folding o f Staphylococcal Nuclease. Biochemistry 1993, 32:2534-2541. The isomerization of Proll7, which is in the cis conformation in the native state, is responsible for the slow-folding kinetic phase observed in wild-type staphylococcal nuclease. Interestingly, a second intermediate kinetic phase is attributed to the isomerization of a non-native c~ proline in the unfolded protein to a native tram proline in the folded protein. The particular proline responsible for this intermediate kinetic phase has not yet been identified.
Result from Proline Cis--Trans Isomerization. Biochemistry 1991, 30:9882-9891.
56. ..
57.
W O O D LC, WHITE I"13, RAMDAS k NAIL BT: Replacement o f a Conserved Proline Eliminates t h e Absorbance-Detected Slow Folding Phase of Iso-2-Cytochrome c Biochemistry 1988, 27:8562-8568.
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HURLE MR, ANDERSON S, KUN'I-Z ID: Confirmation of t h e Predicted Source of a Slow Folding Reaction: Proline 8 of Bovine Pancreatic Trypsin Inhibitor. Protein Eng 1991, 4:451--455.
59.
HERNINGT, YUTAN1K, TANIYAMAY, KmUCHI M: E ects of Proline Mutations o n t h e Unfolding and Refolding of H u m a n Lysozyme: the Slow Refolding Kinetic Phase Does Not
60. •.
RADZICKAA, ACHESON SA, WOLFENDEN R: Cis/Trans Isomerization at Proline: Desolvation a n d its C o n s e q u e n c e s for Protein Folding. Bioorganic Cbem 1992, 20:382-386. A 46-fold rate enhancement is observed for the c~-trans proline isomerization of a small peptide in toluene compared with water. The implication suggested by the authors is that protein folding is more likely to be impeded by proline isomerization reactions involving residues which are solvent exposed in the transition state for refolding. 61. ..
TEXTER FL, SPENCER DB, ROSENSTEIN R, MATTHEWS CR: Intramolecular Catalysis of a Proline Isomerization Reaction in t h e Folding of Dihydrofolate Reductase. Biochemistry 1992, 31:5687-5691. Provides the first mutagenic evidence that a proline isomerization reaction can be intramolecularly catalyzed by an amino acid side chain.
JA Zitzewitz and CR Matthews, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Introduction Site-directed mutagenesis has become a routine method for analyzing the influence of individual amino acid side chains on the structure, function, and stability of proteins [1o]. The information obtained from these studies has been used to re-engineer existing proteins to improve stability or catalytic activity [2°]. Yet the design of entirely new proteins with novel functions remains a major challenge in protein engineering. A serious difficulty in ab initio design is the inability to predict successfully the unique, functional three-dimensional conformation of a protein from its amino acid sequence. Fortunately, protein engineering strategies have also provided new tools to aid in deciphering the rules governing protein folding. The two major impediments to solving the protein folding problem are the high cooperativity and rapidity of the folding react/on. The highly cooperative nature of the folding reaction results in very low equilibrium concentrations of partially folded forms which could provide mechanistic dues. In cases where intermediates are highly populated during the folding reaction, these species often exist for only a few milliseconds. The use of conventional spectroscopic methods to determine their structures and stabilities is simply not feasible. These two obstacles are now being overcome with the advent of several new technologies, including genetic engineering, peptide synthesis, two-dimensional NMR, and stoppedflow circular dichroism (CD). Most of these technological advances have been reviewed recently [3,4oo-6o.]. The purpose of this short review is to survey a number of papers that highlight current advances in protein engineezing strategies for examination of protein folding intermediates. Related issues of protein stability and of fold-
ing and functional modules are discussed in accompanying reviews in this section by Matthews (see pp 589-593) and Landro and Schimmel (see pp 549-554), respectively.
Protein engineering strategies for probing folding intermediates Over 30 years ago, Anfinsen and his colleagues [7] proposed that the linear sequence of amino acids determines the final three-dimensional structure of a protein. A corollary to this hypothesis is that the structure and relative populations of intermediates along the folding pathway, and thus the mechanism of protein folding itself, are also dictated by the primary protein sequence. Thus, changes in the amino acid sequence can potentially alter the structure and the stability of both the final conformation and any folding intermediates. The replacement of particular amino acids in a protein by site-directed mutagenesis offers a unique opportunity to determine the role of these amino acids in the folding process and to test a given protein folding hypothesis. Detailed discussions of such mutagenic analyses can be found in reviews by Matthews [8], Goldenberg [9] and Matouschek and Fersht [10]. Mutagenesis can also be used to incorporate conformational probes at specific sites for studying local folding events. For example, the fluorescence properties of genetically inserted tryptophan probes can be used to characterize protein folding intermediat6s in tryptophanfree proteins [11]. Alternatively, folding can be followed by 19F NMR after growth of tryptophan-containing proreins in the presence of 6-fluorotryptophan [12-]. Mutant proteins which contain unique chemically reac-
Abbreviations BPTI--bovine pancreatic trypsin inhibitor; CD---circular dichroism; PRAI--phosphoribosylanthranilate isomerase.
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Strategies in examining protein folding intermediates Zitzewitz and Matthews tive groups can also be modified to incorporate acceptor/donor pairs for distance measurements in intermediates by fluorescence energy transfer [13]. For example, a cysteine-specific fluorescent probe, such as 5-[ [2-[ (iodoacetyl)amino] ethyl] amino] napthalene 1-sulfonic acid (IAEDANS), can be used as an energy acceptor of donor fluorescence from a single tryptophan residue [14°]. A major challenge of such experiments is to incorporate the probe at a position which does not cause significant disruption of native structure. Cysteine residues, introduced by site-directed mutagenesis, can serve as chemically reactive probes for studying local folding events [15,16]. Probes are introduced into hydrophobic regions which are inaccessible to solvent in the native structure. The chemical accessibility of the cysteine sulfhydryl group to reaction with iodoacetate or 5,5'-dithiobis(2-nitrobenzoate) at different stages of folding can be used, in favorable circumstances, to give a thermodynamic and kinetic description of the folding mechanism in the vicinity of the probe. This method has recently been used to characterize protein folding intermediates in human carbonic anhydrase [17°°] and yeast phosphoglycerate kinase [18.o]. An important control in these experiments is the measurement of the rate of modification of the reactive group in the completely unfolded protein or, preferably, in a peptide containing the cysteine residue. Naturally, the concentration of the reagent and the inherent reactivity must be such that the labeling process occurs more rapidly than subsequent refolding or potential unfolding reactions. Protein fragments can be generated by mutagenesis, either by incorporating new start and stop codons into the structural gene or by introducing specific chemical cleavage sites into the amino acid sequence. Analysis of the equilibrium and kinetic properties for folding of these fragments can identify autonomous folding units and test their role in the folding process [19°]. Care must be taken with this approach in interpretation of the results because the conformations adopted by the fragment may not be the same as those found for the same sequence embedded in the fuR-length protein. For example, the interaction of two or more autonomous folding units may induce further structural changes in the protein. A dramatic example of this phenomenon is the cyclosporin A/cyclophilin system where the cyclosporin A peptide turns inside out upon binding to cyclophilin [20]. Other aspects of protein folding can be studied by using mutagenic methods to make changes in the primary structures of proteins, such as by the Insertion [21] or deletion [22] of loops, or by the construction of disulfide bonds [23°]. More drastic changes, including translocation of the amino and carboxyl termini [24,25], module reshuffling [26] and production of tandemty linked proteins [27°], can also provide new insights into the mechanism by which the sequence guides the folding reaction. Selected protein engineering strategies for studying distinct features of protein folding mechanisms are described in the following sections.
Early intermediates in folding ,. The evidence that has accumulated over the past few years suggests that unfolded proteins (whether unfolding was induced by heat, acid or denaturant) are not completely devoid of residual sttucmre [28]. Recently, Gottfried and Haas [29 "°] measured fluorescence energy transfer between an amino-terminal donor probe and an acceptor probe attached to one of the four lysines of bovine pancreatic trysin inhibitor (BPTI). Their results indicate that BPTI does not exist as a statistical random coil, even under strongly denaturing and reducing conditions. Non-local interactions, responsible for the compact structure in unfolded BPTI, are implicated as essential elements in directing the formation of early folding intermediates. The earliest observable intermediates in protein folding, detected by stopped-flow CD and pulse-labeling NMR, form in less than 5ms and contain substantial nonpolar surfaces and secondary structure [6"']. These very early intermediates are probably an ensemble of conformations in dynamic equilibrium and may contain nonnative elements of structure. Protein fragments may prove useful for kinetic characterization of early intermediates because fragments cannot form the native long-range interactions responsible for supersecondary and tertiary structure formation. In effect, the folding reaction is halted at the stage where all the information contained in the fragment has been processed. Goldberg and colleagues [30o] have recently used stopped-flow CD and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence spectroscopy to show that a carboxy-terminal fragment of the [3 subunit of tryptophan synthase folds in less than 4 ms into a condensed state with non-native secondary structure. The authors conclude that non-specific collapse of a comparable condensed state occurs with the whole protein before reaching the stage at which the first intermediates with native-like secondary structure are observed.
Intermediates that fold directly into the native conformation The rate-limiting step in the folding of many proteins involves the formation of the native conformation. Protein engineering strategies have provided a great deal of insight into the structure of the intermediates that lead directly to the native conformation and the intervening transition states. In many cases, access to this information is enhanced because the rate-limiting step in unfolding is the reverse of the rate-limiting step in refolding. Unfolding is usually a much simpler kinetic process than refolding, making comparison of the properties of the transition state with the native conformation relatively straightforward. The rate-limiting step in the denaturant-induced unfolding and refolding of the at subunit of tryptophan synthase (Fig. 1) involves the interconversion of the native conformation and a stable folding intermediate. This stable intermediate has significant secondary and tertiary structure in the amino folding unit (residues 1-188; 13-strands 1--6)
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Engineering and design but was originally thought to have a disordered carboxy folding unit (residues 189-268; [3-strands 7-8) [31,32]. More recendy, comparisons of single and double mutants at the interface between the folding units have demonstrated the existence of residual structure in the carboxy folding unit, particularly in the vicinity of residues 175 (strand 6) and 209 (strand 7) in the intermediate [33"]. Thus, the final phase in folding appears to involve the docking of strand 8 to strand 1 along with a global repacking of many buried side chains to form the native structure.
Fig. 1. Ribbon diagram of the (x subunit of tryptophan synthase. The eight ~strands that form the core of the barrel are numbered consecutively. The amino folding unit (light grey) comprises 6strands 1-6 and a-helices 0-5, and the carboxy folding unit (dark grey) comprises 13-strands 7-8 and (x-helices 6-8.
Breaking down the cooperativity of the folding reaction A natural consequence of the high cooperativity observed in the folding of globular proteins is the low population of partially folded forms. Mutagenesis provides a potential method for breaking down this cooperativity and for populating folding intermediates. Mutations at the interface between the two folding units of the 0t subunit of tryptophan synthase selectively destabilize the native conformation relative to the stable intermediate [37]. The effect is to enhance the population o f the intermediate in the unfolding transition zone. A similar observation has recently, been made in mutagenic studies invoMng the dimeric protein tryptophan aporepressor (CJ Mann, CA Royer, CR Matthews, unpublished data). Replacements of single tryptophan residues at the dimer interface result in a non-coincidence of the unfolding transitions detected by fluorescence and CD. It appears that these replacements selectively destabilize the interdimer association reaction without greatly perturbing the stability of a folded monomer. The results discussed above, and others [38,39], suggest that the high cooperativity observed for the folding of globular proteins may be a consequence of strong interactions between two or more autonomous folding units. By using mutagenesis to break down the cooperativiw of the folding reaction, folding intermediates can be populated and, thus, a more detailed understanding of the information contained in individual segments of a polypeptide can be obtained. Further insights into how this information is modulated when the segments are annealed in the native conformation can be obtained from additional mutagenic and fragment studies on the structures and stabilities of these partially folded forms.
Disulfide bond rearrangements Fersht and coworkers [34"] have conducted an elaborate mutagenic analysis of the rate-limiting step in the unfolding/refolding pathway of bamase. An extension of the reaction coordinate analysis approach was used to probe the structure of the rate-limiting transition state relative to the native and unfolded forms [35"]. The parameter ¢, introduced to provide a quantitative description of the transition state, equals zero when the transition state is native-like and equals unity when the transition state resembles the unfolded state. Because there is no simple linear relationship between ~ and the extent of structure formation, fractional values of ~ are more difficult to interpret. This type of analysis nevertheless provides useful information about the structure of the rate-limiting transition state for refolding, which, as noted above, is identical to that for unfolding in this instance. The consolidation of the main hydrophobic core, the closing of many loops, and the capping of the amino termini of the helices are the final events in the folding of bamase. Interestingly, a kinetically significant intermediate (or possibly ensemble of intermediates) on the refolding pathway of bamase has a much less stable core than the transition state that leads to the final conformation [36"].
The oxidative folding of reduced BPTI is one of the most extensively studied protein folding mechanisms [40--]. BPTI is a small, single-domain protein which is stabiLized by three disulfide bonds. The rate-limiting step in folding involves a disulfide bond rearrangement prior to the formation of the native structure. Efforts to elucidate the mechanism of folding for BPTI have involved the characterization of partially disulfide-bonded intermediates trapped by iodoacetate during the oxidative refolding of BPTI [41,42]. Protein engineering strategies have been used to synthesize analogs of these trapped intermediates for structural characterization. By replacing cysteines not involved in disulfide bonds with serines or alanines, analogs of the five intermediates retaining one or two of the native disultides were engineered [40 o-]. Four of these analogs, including the (5-55) single-disulfide intermediate [43,44], adopt native-like structures as assessed by NMR spectroscopy [45,46] or X-ray crystallography [47]. An analog of the other crucial single-disulfide intermediate, (30-51), has a native-like [3-sheet and at-helix [48..], as was suggested by earlier studies of a peptide model [49]. Yet the first 14 residues contain little ff any residual structure and residues 37-41 appear to be highly flexible [50"]. Thus,
Strategies in examining protein folding intermediates Zitzewitz and Matthews the (30-51) single-disulfide intermediate may be an example of a true folding intermediate because it contains regions of both native and unfolded structure.
Proline isomerization In 1975, Brandts and his coworkers [51] proposed that the c i s - t r a m isomerization of the peptide bond preceding proline is responsible for the slow refolding of proteins. In this model, only the population of unfolded protein ha~_ng the same isomeric configuration as the native protein is able to fold directly into the native structure. The population of molecules containing non-native proline configurations first has to isomerize its X-Pro (where X represents any amino acid) bonds before folding. Prolines in the c/s conformation in the native protein are often responsible for slow folding reactions (see below). The trans conformation around the peptide bond is expected to be preferred in the unfolded protein based on the predominance of t r a m proline isomers in unstructured peptides [51,52]. Brandts' proline hypothesis is supported by mutagenic studies on several proteins in which the key proline(s) are replaced by other amino acids which isomerize rapidly in the unfolded protein. Replacement of what are often c/s proline residues has led to the loss of a slow refolding phase in several proteins, including thioredoxin [53], ribonuclease T1 [54], ribonuclease A [55 °°] and staphylococcal nuclease [56°°]. Although more difficult to predict, proline isomerization reactions which lead to t r a m prolines in the native structure can also result in slow folding phases, as observed for yeast iso2-cytochrome c [57] and a mutant of BPTI containing only two disulfide bonds [58]. Also, the refolding kinetics of staphylococcal nuclease appears to exhibit phases attributable to both c/s and t r a m configurational isomerization [56"']. Interestingly, a proline-free mutant of human lysozyme exhibits the same denaturant-independent slow refolding kinetic phase observed for the wild-type protein, indicating that this reaction cannot be attributed to proline isomerization [59]. Thus, it is possible that the cis-trans isomerization of other peptide bonds besides those involving prolines may account for some of the slow isomerization reactions observed in protein folding. It should be noted that the significant majority of proline residues in a protein are not involved in rate-limiting isomerization reactions. Wolfenden and coworkers [60..] recently observed that the rate of the c i s - t r a m isomerization of acetyiproline N-methylamide is enhanced in the non-polar solvent toluene. The authors suggest that the isomerization of proline residues buried in the hydrophobic interior of a protein occurs more rapidly than folding reactions. An alternative mechanism for intramolecular catalysis of proline isomerization has been suggested by mutagenic studies of dihydrofolate reductase [61--]. An arginine side chain that is distant in the primary sequence from the critical proline residue, but adjacent to it in the three-dimensional structure, is responsible for this catalysis, presumably by forming
hydrogen bonds with the imide nitrogen, and/or the carbonyl oxygen of the proline. A survey of hydrogenbonding patterns in other proteins reveals a significant number of candidates for this type of acceleration of the proline isomerization reaction. Additional mutagenic studies are required to test the generality of this intramolecular catalysis mechanism.
Conclusion As illustrated by the examples above, protein engineering methodologies provide a very powerful tool for mapping the structure and stability of folding intermediates and their roles in protein folding reactions. Enhanced understanding of critical concepts in protein folding is promised by further applications of protein engineering strategies that combine the tools of recombinant DNA technology with more classical protein chemistry methods. The development of a folding code, analogous to the genetic code, may eventually allow the three-dimensional structure of a protein to be predicted from the linear amino acid sequence. This information would be invaluable for the de novo design of new proteins with novel functions. Furthermore, identification of those sequence features which are critical for successful progression along the folding reaction pathway may help avoid problems of aggregation or proteolysis, ultimately leading to improved yields of products for the biotechnology industry.
Acknowledgements Work from this laboratory is funded by the National Institutes of Health (GM23303) and the National Science Foundation (DMB9004707). JA Zitzewitz is supported by NIH Postdoctoral Fellowship Award GM14954. The authors also kindly thank Deborah Bebout for critical reading of this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •o of outstanding interest 1.
FERSHTA, WINTER G: Protein Engineering. Trends Biochem Sci 1992, 17:292-294. Selected examples of the systematic application of mutagenesis for the analysis of structure, activity, specificity, stability and folding of proteins are highlighted in this short review. In addition, the authors comment on the prospects for protein design from framework architectures or strategies of molecular evolution. .
2. °
RECKTENWALDA, SCHOMBURG D, SCHMID RD: Protein Engineering and Design: Method and Industrial Relevance. J Biotechnol 1993, 28:1-23. This review cites many examples of proteins that have been redestgned to yield improved stability or enzymatic properties and gives a description of the steps involved in the protein design cycle. 3.
K1M PS, BALDWINRI= Intermediates in the Folding Reactions of Small Proteins. A n n u R e v Bic~bem 1990, 59:631-660.
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LECOMTEJTJ, MAr'mEWS CR: Unraveling the Mechanism o f Protein Folding: New Tricks for an Old Problem. Protein Eng 1993, 6:1-10. The application of the recent protein engineering strategies and NMR spectroscopy methods detailed in this review may result in significant progress toward soMng the folding problem during the next few years. 6. MATI'HEWSCR: Pathways o f Protein Folding. Annu Rev .. Bloc,bern 1993, 62:in press. The time-dependent phenomena which occur during protein folding are proposed to involve the progressive development of structure and stability through an ever-slowing series of reactions. 7.
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ROPSONI, FRIEDEN C: Dynamic NMR Spectral Analysis and Protein Folding: Identification of a Highly Populated Folding Intermediate of Rat Intestinal Fatty Acid-Binding Protein by 19F NMR. Proc Natl Acad Sci USA 1992, 89:7222-7226. The localized structure of an intermediate(s) o f rat intestinal fatty acidbinding protein in the vicinity of Trp82 persists at high concentrations of denaturant. This intermediate may be involved in initiating further folding of the ~-sheet. 13.
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JAMESE, WU PG, STITES W, BRAND k Compact Denatured State of a Staphylococcal Nuclease Mutant by Guanidinium as Determined by Resonance Energy Transfer. Biochemistry 1992, 31:10217-10225. A time-resolved resonance energy-transfer study is used to obtain distance distributions in staphylococcal nuclease. The results indicate that residual structure persists in the denatured protein in the presence of concentrations of guamdine hydrochloride of up to 2 M. 15.
BALLERYN, MINARD P, DESMADRILM, BETrON J-M, PERAHIAD, MOUAWADL, HALL L, YON JM: Introduction of Internal Cysteines as Conformational Probes in Yeast Pbosphoglycerate Kinase. Protein Eng 1990, 3:199-204.
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FRESKGARDP-O, C ~ N U, ~ N S S O N L-G, JONSSON B-H: Folding Around the C-Terminus o f Human Carbonic Anhydrase. II: Kinetic Characterization by Use of a Chemically Reactive SH-Group Introduced by Protein Engineering. FEBS Left 1991, 289:117-122.
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MARTENS.SONL-G, JONSSON B-H, FRESKGARDP-O, KIHLGRENA, SVENSSONM, CARLSSONU: Characterization of Folding Intermediates of Human Carbonic Anhydrase If: Probing Substructure by Chemical Labeling of SH Groups Introduced by Site-Directed Mutagenesis. Biochemistry 1993, 32:224-231. The equilibrium folding intermediate of human carbonic anhydrase II has an ordered, native-like secondary structure in the central, twisted ~-sheet, but the peripheral part of the 13-sheet is less ordered as deter-
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BALLERYN, DESMADRILM, MINARDP, Yon JM: Characterization of an Intermediate in the Folding Pathway of Phosphoglycerate Kinase: Chemical Reactivity of Genetically Introduced Cysteinyl Residues During the Folding Process. BiocJoemistry 1993, 32:708-714. The kinetic characterization of a folding intermediate of phosphoglycerate kinase, monitored by accessibility of a unique cysteine residue to chemical reactivity with 5,5'-dithiobis(2-nitrobenzoate) and stoppedflow CD, reveals that the transient intermediate has a high content of secondary structure but a flexible tertiary, structure. 19. ,
EDER J, KIRSCHNER K: Stable Substructures o f EightFold 13cx-Barrel Proteins: Fragment Complementation o f Phosphoribosylanthranilate Isomerase. Biochemistry 1992, 31:3617-3625. Characterization of fragments of phosphoribosyianthranilate isomemse (PRM) supports a folding mechanism involving an intermediate with roughly native-like structure in the first six strands and a partially unfolded structure in the Last two strands. 20.
WLrTHI~CHK, VON FREYBERG B, WEBER C, WIDER G, TRABER R, WIDMER H, BRAUN W: Receptor-Induced Conformation Change o f the lmmunosuppressant Cyclosporin A. Science 1991, 254:953-954.
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23. EDERJ, WILMANNSM: Protein Engineering o f a Disulfide Bond • in a ~/tx-Barrel Protein. Biochemistry 1992, 31:4437---4444. A single disulfide bond is engineered between helices 1 and 8 of PRAI to produce a nearly circular protein with full catalytic activity. The disulfide bond forms spontaneously in vitro and stabilizes the native conformation of the protein without affecting the flexibility of the active site required for catalysis. 24.
LUGERK, HOMMELU, HEROLD M, HOFSTEENGEJ, KIRSCHNERK: Correct Folding of Circularly Permuted Variants of a ~:t Barrel Enzyme in vivo. Science 1989, 243:206-210.
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1WAKURAM, MATrHEWS CR: Construction and Characterization of a Single Polypeptide Chain Containing Two Enzy. matically Active Dihydrofolate Reductase Domains. Protein Eng 1992, 5:791-796. A single polypeptide chain containing two nearly identical dihydrofolate reductase genes is successfully expressed in Eschenchia coli and found to be catalytically active. At low protein concentrations, the denaturantinduced unfolding of the fusion protein is fully reversible and follows a three-state model, suggesting that the two domains fold independently. 28.
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GOTrFRIED DS, HAAS E: Nonlocal Interactions Stabili7e Compact Folding Intermediates in Reduced Unfolded Bovine Pancreatic Trypsin Inhibitor. Biochemistry 1992, 31:12353-12362. Even under strongly reducing and denaturing conditions, BPTI does not exist as a statistical random-coil peptide. The authors propose that the long-range interactions observed in the 'unfolded' protein are involved in coding for the initial steps o f the folding reaction. 30. •
CHAFFOTI'EAF, CADIEUXC, GUILLOUY, GOLDBERGME: A POSsible Initial Folding Intermediate: the C-Terminal Prote-
Strategies in examining protein folding intermediates Z i t z e w i t z a n d M a t t h e w s olytic Domain of Tryptophan Synthase [3 Chain Folds in Less t h a n 4 Milliseconds into a C o n d e n s e d State with Non-Native-Like Secondary Structure. Biochemistry 1992, 31:4303-4308. The non-specific hydrophobic collapse observed for the carboxyterminal fragment of the ~ subunit of tryptophan synthase illustrates the potential usefulness of careful kinetic analyses of fragments for probing folding mechanisms in further detail.
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VAN MIERLO CPM, DARBY NJ, NEUHAUS D, CREIGHTON TE: (14--38,30-51) Double-Disulphide Intermediate in Folding of Bovine Pancreatic TrypsIn Inhibitor: a Two-Dimensional IH Nuclear Magnetic Resonance Study. J Mol Biol 1991, 222:353-371.
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EIGENBROTC, RANDALM, KOSSIAKOFF A& Structural E ects Induced by Removal of a Disulfide-Bridge: t h e X-Ray Structure of t h e C3OA/CS1A Mutant of Basic Pancreatic Trypsin Inhibitor at 1.6A. Protein Eng 1990, 3:591-598.
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TAKASHIT, CHRUNYKBA, CHEN X, MATYHEWSCl~ Mutagenic Analysis of the Interior Packing of an co/J3 Barrel Protein. E ects o n the Stabilities and Rates o f Interconversion of the Native and Partially Folded Forms o f t h e cc Subunit of Tryptophan Synthase. Biochemistry 1993, 32:5566-5575. The mutagenic studies presented in this paper reveal that the stable intermediate of the ct subunit of tryptophan synthase that is involved in the rate-limiting conversion to the native conformation contains some organized structure in the carboxy folding unit. The general implication for folding is that more stable substructures form first and then shift the equilibrium of less stable elements toward the folded form, leading to the progressive development of native-like structure. 34. •
SERRANOL MATOUSCHEKA, FERSHT AR: T h e Folding o f an Enzyme. III. Structure of t h e Transition State for Unfolding of Barnase Analyzed by a Protein Engineering Procedure. J Mol Biol 1992, 224:805-818. A thorough study of more than 50 mutations provides a detailed description of the first transition state in the unfolding pathway of barrlase. 35. •.
FERSHT AR, MATOUSCHEK A, SERRANO L: T h e Folding of an Enzyme. I. Theory of Protein Engineering Analysis of Stability and Pathway of Protein Folding. J Mol Biol 1992, 224:771-782. The theoretical analysis of a protein engineering approach for mapping the formation of structure in transition states and folding intermediates, based on an extended reaction coordinate analysis approach, is presented in this paper. 36. •
MATOUSCHEKA, SERRANO L, FERSHT AI~ T h e Folding o f an Enzyme. IV. Structure of an Intermediate In t h e Refolding of Barnase Analyzed by a ProteIn Engineering Procedure. J Mol Biol 1992, 224:819--835. The analysis of over 40 mutations is used to detail carefully the regions of structure in a kinetic intermediate(s) on the folding pathway of barnase. It appears that although the intermediate has a significant amount of structure, it has a less stable core than in the transition state leading to native protein. 37.
CHEN X, RAMBO R, MATrHEWS CR~ Amino Acid Replacemerits Can Selectively A ect the Interaction Energy of A u t o n o m o u s Folding Units in the ~x Subunit of Tryptophan Synthase. Biochemistry 1992, 31:2219-2223.
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vAN MIERLO CPM, DARBY NJ, CREIGHTON TE: The Partially Folded Conformation of the Cys30-Cys51 Intermediate in t h e Disulfide Folding Pathway of Bovine Pancreatic Trypsin Inhibitor. Proc Natl Acad Sci USA 1992, 89:6775-6779. An analog of the single disulfide-bonded intermediate (30-51) of BPTI, containing serine replacements for the other four cysteines, has a nativelike secondary structure in the core and a disordered amino terminus. Thus, the intermediate reflected by this analog may be the only well populated disulfide-bonded intermediate in the folding pathway of BPTI which is a true folding intermediate.
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vAN MIERLO CPM, DARBYNJ, KEELERJ, NEUHAUSD, CREIGHTON TE: Partially Folded Conformations o f t h e ( 3 0 - 5 1 ) Intermediate in t h e Disulphide Folding Pathway of Bovine Pancreatic Trypsin Inhibitor. tH and 15N Resonance Assignm e n t s and Determination of Backbone Dynamics from 15N Relaxation Measurements. J Mol Biol 1993, 229:1125-1146. Residues 1-14 and 37-41 are highly flexible in this (30-51) folding intermediate of BPTI. This flexibility may explain why any of the possible disulfide bonds involving Cys5, Cysl4 or Cys38 can be formed from this intermediate during folding.
•
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55. ••
SCHULTZDA, SCHMID FX, BALDWINRL: Cis Proline Mutants of Ribonuclease A. II. Elimination of t h e Slow-Folding Forms by Mutation. Protein Sci 1992, 1:917-924. O n the basis of a careful kinetic analysis of the unfolding/refolding of single- and double-proline mutants, the authors conclude that prolines 93 and 114, the two cis prolines in the native conformation of ribonuclease A, are jointly, responsible for forming the major slow-folding intermediate
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Engineering and design NAKANOT, ANTONINO LC, FOX RO, FINK A L E ect of Proline Mutations o n t h e Stability a n d Kinetics of Folding o f Staphylococcal Nuclease. Biochemistry 1993, 32:2534-2541. The isomerization of Proll7, which is in the cis conformation in the native state, is responsible for the slow-folding kinetic phase observed in wild-type staphylococcal nuclease. Interestingly, a second intermediate kinetic phase is attributed to the isomerization of a non-native c~ proline in the unfolded protein to a native tram proline in the folded protein. The particular proline responsible for this intermediate kinetic phase has not yet been identified.
Result from Proline Cis--Trans Isomerization. Biochemistry 1991, 30:9882-9891.
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W O O D LC, WHITE I"13, RAMDAS k NAIL BT: Replacement o f a Conserved Proline Eliminates t h e Absorbance-Detected Slow Folding Phase of Iso-2-Cytochrome c Biochemistry 1988, 27:8562-8568.
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60. •.
RADZICKAA, ACHESON SA, WOLFENDEN R: Cis/Trans Isomerization at Proline: Desolvation a n d its C o n s e q u e n c e s for Protein Folding. Bioorganic Cbem 1992, 20:382-386. A 46-fold rate enhancement is observed for the c~-trans proline isomerization of a small peptide in toluene compared with water. The implication suggested by the authors is that protein folding is more likely to be impeded by proline isomerization reactions involving residues which are solvent exposed in the transition state for refolding. 61. ..
TEXTER FL, SPENCER DB, ROSENSTEIN R, MATTHEWS CR: Intramolecular Catalysis of a Proline Isomerization Reaction in t h e Folding of Dihydrofolate Reductase. Biochemistry 1992, 31:5687-5691. Provides the first mutagenic evidence that a proline isomerization reaction can be intramolecularly catalyzed by an amino acid side chain.
JA Zitzewitz and CR Matthews, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.