Hierarchical unfolding of the α-lactalbumin molten globule: presence of a compact intermediate without a unique tertiary fold1

doi:10.1006/jmbi.2000.3660 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 298, 1±6

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Hierarchical Unfolding of the a -Lactalbumin Molten Globule: Presence of a Compact Intermediate Without a Unique Tertiary Fold Suroopa Chakraborty and Zheng-yu Peng* Department of Biochemistry University of Connecticut Health Center, 263 Farmington Avenue, Farmington CT 06030, USA

The difference between the framework model and the hydrophobic collapse model of protein folding largely rests on whether a secondarystructure framework can exist independently of native tertiary interactions. Here, we used circular dichroism and disul®de exchange experiments to examine the unfolding mechanism of a-LA(a), a twodisul®de variant of human a-lactalbumin (a-LA) that adopts a molten globule conformation under near physiological conditions. Our results show that as the concentration of denaturant increases, the a-LA molten globule ®rst loses its ability to form a speci®c, native-like tertiary fold. Subsequently, at a higher denaturant concentration, the protein loses its secondary structure and adopts an extended conformation. A compact, non-native disul®de bond isomer, which does not form signi®cantly under both native and strongly denaturing conditions, was found to be moderately populated in 2 M guanidine hydrochloride (GuHCl). Qualitatively the same result was also obtained in urea. These results suggest that formation of secondary structure is a necessary, but not suf®cient condition for formation of the native-like tertiary fold and support a hierarchical model of protein folding. # 2000 Academic Press

*Corresponding author

Keywords: a-lactalbumin; molten globule; circular dichroism; disul®de exchange; protein folding

Two models have been commonly used to describe the process of spontaneous protein folding (for reviews, see Baldwin & Rose, 1999a,b). In the framework model, the folding is initiated by formation of local secondary structure elements. These nascent elements diffuse in solution, collide and coalesce into a native-like tertiary fold, which subsequently ®xes the side chain packing interactions (Kim & Baldwin, 1982; Ptitsyn, 1973). In the hydrophobic collapse model, formation of secondary and tertiary structures occurs simultaneously and is driven by the same process, that is the burial of hydrophobic residues (Dill, 1985, 1990). Distinguishing these two models is not Abbreviations used: a-LA, a-lactalbumin; a-LA(a), recombinant human a-LA with C61, C73, C77, and C91 replaced by an alanine residue and an additional Nterminal methionine residue; CD, circular dichroism; GuHCl, guanidine hydrochloride. E-mail address of the corresponding author: [email protected] 0022-2836/00/010001±6 $35.00/0

always easy. Although for many proteins, formation of secondary structure precedes the formation of a rigid, speci®c tertiary structure, it is still possible that a native-like tertiary fold or backbone topology forms at a very early stage of folding. For example, a native-like tertiary fold was observed in the molten globule form of a-lactalbumin (a-LA) and apo-myoglobin (Peng & Kim, 1994; Rischel et al., 1996; Wu et al., 1995). This equilibrium partially folded state resembles the early kinetic folding intermediate of these proteins (Ikeguchi et al., 1986; Jennings & Wright, 1993; Kuwajima et al., 1985). Fluorescence energy transfer studies showed that a native-like distance between Trp59 and the heme group in cytochrome c and between the two domains in adenylate kinase are reached in millisecond time scales, which can be interpreted as formation of a nativelike tertiary topology (Elove et al., 1992; Shastry & Roder, 1998). On the other hand, peptide models corresponding to isolated secondary structure elements are often unfolded in aqueous solution, # 2000 Academic Press

2 precluding the study of their intrinsic stability. Therefore, it is still unclear whether a secondary structure framework can exist independent of native-like tertiary interactions. One way to circumvent some of these dif®culties is to study the folding process at equilibrium. In these studies, emphasis is not placed on the explicit time dependence of the folding reaction. Instead, the structural property of a protein is monitored as a function of the free energy difference between the folded and unfolded state (Shortle et al., 1996). This strategy is particularly useful when the protein can exist in several partially folded conformations. An advantage of this approach is that if an equilibrium folding intermediate is observed, it must correspond to the global free energy minimum under that condition, while a kinetic folding intermediate can arise from a local free energy minimum that acts as a kinetic trap. a-LA is a small, two-domain protein that adopts a partially folded structure, often referred to as the molten globule, under a variety of mildly denaturing conditions (for reviews, see Arai & Kuwajima, 2000; Ptitsyn, 1996). Here, we examine the unfolding of a-LA molten globule for two reasons. First, it appears to be an obligatory intermediate for a-LA folding (Arai & Kuwajima, 1996; Kuwajima et al., 1989). Second, it has a native-like tertiary topology, mainly localized in the a-helical domain, in addition to high levels of secondary structure and a compact geometry (Peng & Kim, 1994; Wu et al., 1995). These observations suggest that a signi®cant portion of the information transfer process from one dimensional amino acid sequence to three dimensional structure is completed at the molten globule stage of folding. Thus, it is important to understand the mechanism for native tertiary fold formation in the a-LA molten globule. Figure 1(a) shows the X-ray crystal structure of a-LA. In an earlier study, we have constructed a variant of human a-LA, called a-LA(a), that contains four cysteine residues in the a-helical domain, with the remaining cysteine residues replaced by alanine (Wu et al., 1995, 1996). The a-LA(a) does not fold into a rigid structure; instead, it remains in a molten globule-like conformation even at neutral pH in absence of denaturant. The four cysteine residues in a-LA(a) can form three disul®de bonded isomers (Figure 1(b)). Under native conditions, a-LA(a) prefers to form the two native disul®de bonds, (28-111) and (6-120), suggesting that a native-like tertiary fold has the lowest free energy. Under strongly denaturing conditions, a-LA(a) prefers to form two non-native disul®de bonds, (6-28) and (111-120). These disul®de bonds are between cysteine residues that are the nearest neighbors in the primary sequence, suggesting that under these conditions, the polypeptide behaves like a random polymer (Wu et al., 1995). To investigate the equilibrium unfolding process of a-LA(a), we performed disul®de exchange stu-

Hierarchical Unfolding of the -LA Molten Globule

Figure 1. (a) The structure of human a-LA (Acharya et al., 1991). The a-helical domain (shaded) contains essentially all a-helical secondary structure, whereas the b-sheet domain contains a small, antiparallel b-sheet and several loop-like structures. The disul®de bonds are shown as the ball-and-stick model and labeled with residue numbers of cysteines they connect. In a-LA(a), Cys61, Cys73, and Cys77 in the b-sheet domain and Cys91 which participates in an inter-domain disul®de bond are replaced by alanine. The structure was produced using the program MOLMOL (Koradi et al., 1996) and PDB coordinate 1HML. (b) There are three possible con®gurations for the four cysteine residues in a-LA(a) to form two disul®de bonds. (6-120; 28-111) contains the two native (naturally occurring) disul®de bonds which form preferentially under native conditions (Wu et al., 1995). (6-28; 111-120) contains two non-native disul®de bonds between cysteine residues that are the nearest neighbors in the primary sequence. This disul®de bond isomer forms preferentially under strongly denaturing conditions. (6-111; 28-120) contains two long-range, nonnative disul®de bonds which do not form signi®cantly under both native and strongly denaturing conditions.

dies as a function of guanidine hydrochloride (GuHCl) concentration (Figure 2). Surprisingly, the disul®de bond isomer, (6-111; 28-120), which does not form signi®cantly under both native and strongly denaturing conditions, was found to be moderately populated in low concentrations of GuHCl. The maximum amount of (6-111; 28-120), which reaches 20 % of the total two-disul®de bond species, was observed in 2 M GuHCl. After that, it decays monotonically. Formation of different disul®de bond isomers indicates that the protein no longer has a unique, speci®c tertiary fold. Since the isomer (6-111; 28-120), like the native

Hierarchical Unfolding of the -LA Molten Globule

3

Figure 2. HPLC analysis of the disul®de exchange mixtures obtained in (a) 0 M, (b) 2 M, and (c) 6 M GuHCl at 20  C. The results obtained in 0 and 6 M GuHCl are qualitatively similar to that of Wu et al. (1995), except for some small differences which could arise for the following two reasons. First, we used 2 mM protein concentration whereas the previous investigators used 5 mM protein concentration. Second, the current experiments were carried out at a slightly lower temperature (20  C). The previous experiments were carried out at the ambient temperature of the anaerobic chamber, which was typically a few degrees higher than the room temperature. The identity of three two-disul®de bond isomers are labeled according to the assignment of Wu et al. (1995). Other HPLC peaks correspond to mixed disul®de bond species between a-LA(a) and glutathione; their presence does not affect the ratio between the three two-disul®de bond isomers. a-LA(a) was expressed and puri®ed from E. coli as described previously (Peng & Kim, 1994; Wu et al., 1995). The recombinant protein carries an additional N-terminal methionine. In the native state, this extra residue reduces the thermal stability and calcium binding af®nity of a-LA, although the structures obtained with and without the N-terminal methionine are essentially identical (Chaudhuri et al., 1999; Ishikawa et al., 1998; Veprintsev et al., 1999). The disul®de exchange mixture consisted of 2 mM protein in buffers containing 10 mM Tris (pH 8.5), 1 mM EDTA, with various amount of GuHCl. To this solution, 100 mM of reduced glutathione and 10 mM of oxidized glutathione (®nal concentrations) were added to catalyze the reaction. The samples were incubated for 16 to 24 hours at 20  C in a refrigerated water bath installed inside the anaerobic chamber with an oxygen partial pressure less than 1 ppm. The attainment of equilibrium was con®rmed by varying the incubation time (from 4 to 16 hours, the equilibrium was typically reached in about eight hours). The disul®de exchange reactions were quenched by adding an equal amount of 10 % acetic acid and the mixtures were loaded onto a Vydac C18 analytical column eluted with a gradient from 38 to 47 % acetonitrile in water containing 0.1 % TFA. The total length of the gradient was 100 min.

disul®de bond isomer (6-120; 28-111), contains two long-range disul®de bonds between cysteines distant in the primary sequence, our data suggest that during the denaturation process, the a-LA molten globule ®rst loses its ability to distinguish the native-like tertiary fold from other compact, nonnative folds, which occurs before the molecule adopts a random, extended conformation. In order to see if the formation of (6-111; 28120) is an intrinsic property of the a-LA molecule or is due to speci®c interactions between a-LA and GuHCl, we compared the disul®de exchange pattern obtained in GuHCl with that obtained in urea (Figure 3(a) and (b)). Qualitatively the same features were observed with both denaturants, although the maximum amount of (6-111; 28-120) observed in the urea-induced denaturation was somewhat less than that

observed in the GuHCl-induced denaturation (Figure 3(c)). In addition, an isolated a-helical domain of a-LA, called a-Domain (Peng & Kim, 1994), also exhibits the same behavior (data not shown). Taken together, these results suggest that the formation of the non-speci®c disul®de isomer, (6-111; 28-120), is an intrinsic property of the a-helical domain of a-LA. It is particularly interesting to compare the loss of speci®city for formation of the native-like tertiary fold with the disruption of secondary structure during the process of unfolding. Figure 3(d) shows the urea-induced denaturation of a-LA(a), as monitored by the a-helical circular dichroism (CD) signal at 222 nm. Clearly, formation of the non-speci®c disul®de isomer (6-111; 28-120) occurs at a lower denaturant concentration than disruption of secondary structure, even when the disul-

4

Hierarchical Unfolding of the -LA Molten Globule

Figure 3. (a) The percentage of (6-120; 28-111) (open circles), (6111; 28-120) (®lled circles), and (628; 111-120) (open squares) in the total two-disul®de bond isomer population as a function of GuHCl concentration. The percentage was calculated using the following formula: Pi ˆ ai/(a(6-28; 111-120) ‡ a(6-111; 28-120) ‡ a(6-120; 28-111)), where ai is the amount of each two-disul®de bond isomer as determined by integration of the HPLC chromatograms. (b) Same as (a), except urea, instead of GuHCl, was used as a denaturant. (c) Comparison of the amount of the non-speci®c disul®de intermediate, (6-111; 28-120), as a function of denaturant concentration for both GuHCl (open circles) and urea (®lled circles). For each denaturant concentration, the experiment has been repeated two to three times. The standard deviation was indicated in the graph. (d) The urea-induced denaturation curve of oxidized a-LA(a) with two native disul®de bonds (6-120; 28-111) (open circles) and reduced a-LA(a) (®lled circles) at 20  C. The samples consisted of 10 mM protein in buffers containing 10 mM Tris (pH 8.5), 1 mM EDTA, with various amount of urea. For reduced a-LA(a), the buffer also contained 1 mM DTT. At each urea concentration, the CD signal at 222 nm was averaged for ®ve minutes using a JASCO J-715 spectropolarimeter equipped with a thermoelectric temperature controller and a 1 mm pathlength cuvette. In Figure 3(a), (b) and (c), smooth lines were drawn to guide the reader; these lines have no theoretical meaning. In (d), the lines represent the best ®t using a two-state unfolding model.

®de bonds in a-LA(a) are reduced. As noted previously, the maximum amount of (6-111; 28-120) was obtained in 2 M GuHCl or urea (Figure 3(c)). In contrast, the mid-point of secondary structure unfolding occurs at 3.5 M urea for reduced a-LA(a) and 5 M urea for a-LA(a) with the two native disul®de bonds{. Thus, the formation of secondary structure and formation of the nativelike tertiary fold are apparently driven by different molecular interactions. Indeed, the loss of a-helical secondary structure in reduced a-LA(a) approximately coincides with the formation of disul®de bond isomer (6-28; 111-120) (c.f. Figure 3(b) and (d)), suggesting that disruption of secondary structure is correlated with formation of the extended molecule. Based on the disul®de exchange data, the unfolding of a-LA(a), a molten globule form of a-LA, clearly is not two-state. This observation is consistent with previous proline scanning mutagenesis and NMR studies (Schulman & Kim, 1996; Schulman et al., 1997). However, the urea unfolding curve of a-LA(a) alone, as monitored by CD, can be ®t with an apparent two-state transition. Recently, it has been shown that the presence of the 28-111 disul®de bond constrains { The GuHCl-induced denaturation of reduced aLA(a), as monitored by CD at 222 nm, does not exhibit a sinusoidal transition, perhaps because GuHCl acts as a salt.

the structure of the a-LA molten globule and weakens its folding cooperativity (Luo & Baldwin, 1999). In our opinion, this cannot fully explain the formation of (6-111; 28-120), since during the disul®de exchange reaction, the disul®de bonds in a-LA(a) are allowed to break and reform, therefore, they serve more as a reporter rather than as a constraint to the molecular structure. It is more likely that the non-cooperative folding can be detected by certain methods, but not by others. In this regard, it is interesting to note that the folding of all-Ala a-LA, which does not contain any disul®de bond, still exhibits some non-cooperative character (Red®eld et al., 1999). Our results suggest that the folding of a-LA molten globule can be described by a hierarchical, framework-like model. In this model, formation of secondary structure occurs prior to formation of the native-like tertiary topology. There is one signi®cant difference between this model and the traditional framework model, that is in this model, the secondary structure formation and formation of a compact state occur simultaneously (Chan & Dill, 1990). In this respect, it is reminiscent to the hydrophobic collapse model. The early-formed secondary structure elements in a-LA molten globule may be stabilized by local interactions, such as a-helical propensities, or by non-local hydrophobic effects. These interactions, however, are not suf®cient to specify a unique, native-like tertiary fold. As the

Hierarchical Unfolding of the -LA Molten Globule

folding process precedes, the a-LA molten globule may acquire additional long-range interactions, between secondary structure elements that have already formed. This explains why the folding process of a-LA molten globule appears to be non-cooperative. Whether a protein will fold or unfold according to such a hierarchical model may depend on the relative stability of secondary structure and tertiary interactions. Previous studies of peptide fragments corresponding to the isolated a-helices in a-LA and lysozyme suggested that these molecules are typically unstructured in solution (Demarest et al., 1998, 1999; Shimizu et al., 1996; Yang et al., 1995). However, some of these peptides can fold into an a-helical conformation when interacting with another peptide, even though the interaction may not be native-like (Demarest et al., 1999; Vuilleumier & Mutter, 1993; Peng, Z.-Y. unpublished observations). Thus, a secondary structure framework can exist in a protein-like environment independent of native tertiary interactions. In the a-LA molten globule, substitutions of an a-helical residue by proline, which disrupts the a-helices, typically have a detrimental effect on both secondary structure and the native tertiary topology (Schulman & Kim, 1996). On the other hand, substitutions of a buried hydrophobic residue by alanine, which has a high a-helical propensity, often affect the ability to form the native-like tertiary topology but not secondary structure (Song et al., 1998; Wu & Kim, 1998). Taken together, these results suggest that formation of secondary structure is a necessary, but not suf®cient condition for formation of the native-like tertiary fold.

Acknowledgements This work was supported by a grant from the National Institute of Health (GM-54533). Z.-Y. P. thanks Peter Kim, in whose lab this investigation was initiated. The authors thank Yongzhang Luo, Brenda Schulman, and Elisha Haas for communication of preliminary results and Dan Minor, Leila Mosavi, and Peter Setlow for critical reading of the manuscript.

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Edited by C. R. Matthews (Received 1 November 1999; received in revised form 3 February 2000; accepted 25 February 2000)