The mechanism of beta-hairpin formation

Beta-hairpins constitute an important class of connecting protein secondary structures. Several groups have postulated that such structures form early in the folding process and serve to nucleate the formation of extended beta-sheet structures. Despite the importance of beta-hairpins in protein folding, little is known about the mechanism of formation of these structures. While it is well established that there is a complex interplay between the stability of a beta-hairpin and loop conformational propensity, loop length, and the formation of stabilizing cross-strand interactions (H-bonds and hydrophobic interactions), the influence of these factors on the folding rate is poorly understood. Peptide models provide a simple framework for exploring the molecular details of the formation of beta-hairpin structures. We have explored the fundamental processes of folding in two linear peptides that form beta-hairpin structures, having a stabilizing hydrophobic cluster connected by loops of differing lengths. This approach allows us to evaluate existing models of the mechanism of beta-hairpin formation. We find a substantial acceleration of the folding rate when the connecting loop is made shorter (i.e., the hydrophobic cluster is moved closer to the turn). Analysis of the folding kinetics of these two peptides reveals that this acceleration is a direct consequence of the reduced entropic cost of the smaller loop search.     

The folding of an enzyme. VI. The folding pathway of barnase: comparison with theoretical models.

The sequence of events in the refolding pathway of barnase has been analysed to search for general principles in protein folding. There appears to be a correlation between burying hydrophobic surface area and early folding events. All the regions that fold early interact extensively with the beta-sheet. These interactions involve predominantly hydrophobic interactions and the burial of very extensive hydrophobic areas in which multiple, close, hydrophobic-hydrophobic contacts are established around a central group of aliphatic residues. There is no burial of hydrophilic residues in these regions; those that are partly screened from the solvent make hydrogen bonds. All the regions or interactions that are made late in the folding pathway do not make extensive contacts with the beta-sheet. Their buried hydrophobic regions lack a central hydrophobic residue or residues around which other hydrophobic residues pack. Further, in some of these regions there is an extensive burial of hydrophilic residues. The results are consistent with one of the earlier events in protein folding being the local formation of native-like secondary structure elements driven by local hydrophobic surface burial. A possible candidate for an initiation site is a beta-hairpin between beta-strands 3 and 4 that is conserved in the microbial ribonuclease family. A comparison of structures in this family shows that those regions that can be superimposed, or have sequence homology, correspond to elements of structure that are formed and interact with each other early in the folding pathway, suggesting that some of these residues could be involved in directing the folding process. The data on barnase combined with results from other laboratories suggest the following tentative conclusions for the refolding of small monomeric proteins. (1) The refolding pathway is, at least in part, sequential and of compulsory order. (2) Secondary structure formation is driven by local hydrophobic surface burial and precedes the formation of most tertiary interactions. These elements are then stabilized and sometimes elongated by tertiary interactions. It is plausible that there are stop signals encoded in the linear sequence that prevent the elongation of isolated secondary structure elements in solution to a larger extent than is found in the folded protein. (3) Many tertiary interactions are not very constrained in the intermediate but become more and more defined as the hydrophobic cores consolidate, loop structures form and the configuration of surface residues takes place. The interactions between different elements of secondary structure are the last ones to be consolidated while the interactions within the secondary structure elements are consolidated earlier.(ABSTRACT TRUNCATED AT 400 WORDS)     

The chicken-egg scenario of protein folding revisited

What is the first step in protein folding - hydrophobic collapse (compaction) or secondary structure formation? It is still not clear if the major driving force in protein folding is hydrogen bonding or hydrophobic interactions or both. We analyzed data on the conformational characteristics of 41 globular proteins in native and partially folded conformational states. Our analysis shows that a good correlation exists between relative decrease in hydrodynamic volume and increase in secondary structure content. No compact equilibrium intermediates lacking secondary structure, or highly ordered non-compact species, were found. This correlation provides experimental support for the hypothesis that hydrophobic collapse occurs simultaneously with formation of secondary structure in the early stages of the protein folding.     

Fundamental processes of protein folding: measuring the energetic balance between helix formation and hydrophobic interactions

Theories of protein folding often consider contributions from three fundamental elements: loops, hydrophobic interactions, and secondary structures. The pathway of protein folding, the rate of folding, and the final folded structure should be predictable if the energetic contributions to folding of these fundamental factors were properly understood. alphatalpha is a helix-turn-helix peptide that was developed by de novo design to provide a model system for the study of these important elements of protein folding. Hydrogen exchange experiments were performed on selectively 15N-labeled alphatalpha and used to calculate the stability of hydrogen bonds within the peptide. The resulting pattern of hydrogen bond stability was analyzed using a version of Lifson-Roig model that was extended to include a statistical parameter for tertiary interactions. This parameter, x, represents the additional statistical weight conferred upon a helical state by a tertiary contact. The hydrogen exchange data is most closely fit by the XHC model with an x parameter of 9.25. Thus the statistical weight of a hydrophobic tertiary contact is approximately 5.8x the statistical weight for helix formation by alanine. The value for the x parameter derived from this study should provide a basis for the understanding of the relationship between hydrophobic cluster formation and secondary structure formation during the early stages of protein folding.     

Nonadditivity in conformational entropy upon molecular rigidification reveals a universal mechanism affecting folding cooperativity

Previously, we employed a Maxwell counting distance constraint model (McDCM) to describe α-helix formation in polypeptides. Unlike classical helix-coil transition theories, the folding mechanism derives from nonadditivity in conformational entropy caused by rigidification of molecular structure as intramolecular cross-linking interactions form along the backbone. For example, when a hydrogen bond forms within a flexible region, both energy and conformational entropy decrease. However, no conformational entropy is lost when the region is already rigid because atomic motions are not constrained further. Unlike classical zipper models, the same mechanism also describes a coil-to-β-hairpin transition. Special topological features of the helix and hairpin structures allow the McDCM to be solved exactly. Taking full advantage of the fact that Maxwell constraint counting is a mean field approximation applied to the distribution of cross-linking interactions, we present an exact transfer matrix method that does not require any special topological feature. Upon application of the model to proteins, cooperativity within the folding transition is yet again appropriately described. Notwithstanding other contributing factors such as the hydrophobic effect, this simple model identifies a universal mechanism for cooperativity within polypeptide and protein-folding transitions, and it elucidates scaling laws describing hydrogen-bond patterns observed in secondary structure. In particular, the native state should have roughly twice as many constraints as there are degrees of freedom in the coil state to ensure high fidelity in two-state folding cooperativity, which is empirically observed.     

Transition states for protein folding have native topologies despite high structural variability

We present a structural analysis of the folding transition states of three SH3 domains. Our results reveal that the secondary structure is not yet fully formed at this stage of folding and that the solvent is only partially excluded from the interior of the protein. Comparison of the members of the transition state ensemble with a database of native folds shows that, despite substantial local variability, the transition state structures can all be classified as having the topology characteristic of an SH3 domain. Our results suggest a mechanism for folding in which the formation of a network of interactions among a subset of hydrophobic residues ensures that the native topology is generated. Such a mechanism enables high fidelity in folding while minimizing the need to establish a large number of specific interactions in the conformational search.     

Mechanism of protein folding II. Lysozyme and phospholipase

Refolding of hen egg-white lysozyme assuming the formation of secondary structures (-helices and β-sheets) is carried out by the method presented in the previous paper (N. Saitô et al., Proteins; Struct. Funct. Genet. 3 (1988) 199–208). To do this, the hydrophobic interactions between the hydrophobic residues which are located at the key positions for folding and can be identified without te knowledge of the native structure, and the nonbonded interactions between every pair of atoms (except hydrogen) or groups are introduced successively from short-to medium-distance pairs. The search for the energy minimum by these interactions can afford a conformation of especially the mutual arrangements between neighboring secondary structures. When these local structures are accomplished, some of the long-distance amino-acid pairs come close together and then the possible interactions (hydrophobic, nonbonded) are introduced. The three-dimensional structure of lysozyme thus obtained is shown to have locally correct arrangements of the secondary structures, but mutual relations between long-distance parts of the chain are not similar to the native structure. The introduction of disulfide bonds between appropriate cysteine residues is necessary to reach the native structure. The choice of cysteine pairs for disulfide bonding is made by the criterion given in the paper to follow (K. Watanabe, A. Nakamura, Y. Fukuda and N. Saitô, Biophys. Chem. 40 (1991) 293). The same treatment is applied to bovine pancreatic phospholipase with 7 disulfide bonds. The formation of the antiparallel β-structures from neighboring β-strands and the problem of the folding order are also discussed.     

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Trifluoroethanol may form a solvent matrix for assisted hydrophobic interactions between peptide side chains

Several models for interactions between trifluoroethanol (TFE) and peptides and proteins have recently been proposed, but none have been able to rationalize the puzzling observations that on the one hand TFE can stabilize some hydrophobic interactions in secondary structures, but on the other can also melt the hydrophobic cores of globular proteins. The former is illustrated in this paper by the effect of TFE on a short elastin peptide, GVG(VPGVG)(3), which forms type II beta-turns stabilized by hydrophobic interactions between two intra-turn valine side chains. This folding, driven by increasing the entropy of bulk water, is stimulated in TFE-water mixtures and/or by raising the temperature. To explain these apparently contradictory observations, we propose a model in which TFE clusters locally assist the folding of secondary structures by first breaking down interfacial water molecules on the peptide and then providing a solvent matrix for further side chain--side chain interactions. This model also provides an explanation for TFE-induced transitions between secondary structures, in which the TFE clusters may redirect non-local to local interactions.     

Solvent effects on the conformational transition of a model polyalanine peptide

We have investigated the folding of polyalanine by combining discontinuous molecular dynamics simulation with our newly developed off-lattice intermediate-resolution protein model. The thermodynamics of a system containing a single Ac-KA(14)K-NH(2) molecule has been explored by using the replica exchange simulation method to map out the conformational transitions as a function of temperature. We have also explored the influence of solvent type on the folding process by varying the relative strength of the side-chain's hydrophobic interactions and backbone hydrogen bonding interactions. The peptide in our simulations tends to mimic real polyalanine in that it can exist in three distinct structural states: alpha-helix, beta-structures (including beta-hairpin and beta-sheet-like structures), and random coil, depending upon the solvent conditions. At low values of the hydrophobic interaction strength between nonpolar side-chains, the polyalanine peptide undergoes a relatively sharp transition between an alpha-helical conformation at low temperatures and a random-coil conformation at high temperatures. As the hydrophobic interaction strength increases, this transition shifts to higher temperatures. Increasing the hydrophobic interaction strength even further induces a second transition to a beta-hairpin, resulting in an alpha-helical conformation at low temperatures, a beta-hairpin at intermediate temperatures, and a random coil at high temperatures. At very high values of the hydrophobic interaction strength, polyalanines become beta-hairpins and beta-sheet-like structures at low temperatures and random coils at high temperatures. This study of the folding of a single polyalanine-based peptide sets the stage for a study of polyalanine aggregation in a forthcoming paper.     

The sequence TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase displays structural ambivalence and interconverts between alpha-helical and beta-hairpin conformations mediated by collapsed conformational states.

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.     

Involvement of electrostatic interactions in the mechanism of peptide folding induced by sodium dodecyl sulfate binding

Sodium dodecyl sulfate (SDS) has consistently been shown to induce secondary structure, particularly alpha-helices, in polypeptides, and is commonly used to model membrane and other hydrophobic environments. However, the precise mechanism by which SDS induces these conformational changes remains unclear. To examine the role of electrostatic interactions in this mechanism, we have designed two hydrophilic, charged amphipathic alpha-helical peptides, one basic (QAPAYKKAAKKLAES) and the other acidic (QAPAYEEAAEELAKS), and their structures were studied by CD and NMR. The design of the peptides is based on the sequence of the segment of residues 56-70 of human platelet factor 4 [PF4(56-70), QAPLYKKIIKKLLES]. Both peptides were unstructured in water, and in the presence of neutral, zwitterionic, or cationic detergents. However, in SDS at neutral pH, the basic peptide folded into an alpha-helix. By contrast, the pH needed to be lowered to 1.8 before alpha-helix formation was observed for the acidic peptide. Strong, attractive electrostatic interactions, between the anionic groups of SDS and the cationic groups of the lysines, appeared to be necessary to initiate the folding of the basic peptide. NMR analysis showed that the basic peptide was fully embedded in SDS-peptide micelles, and that its three-dimensional alpha-helical structure could be superimposed on that of the native structure of PF4(56-70). These results enabled us to propose a working model of the basic peptide-SDS complex, and a mechanism for SDS-induced alpha-helical folding. This study demonstrates that, while the folding of peptides is mostly driven by hydrophobic effects, electrostatic interactions play a significant role in the formation and the stabilization of SDS-induced structure.     

Ultraviolet Raman examination of the environmental dependence of bombolitin I and bombolitin III secondary structure.

Bombolitin I and III (BI and BIII) are small amphiphilic peptides isolated from bumblebee venom. Although they exist in predominately nonhelical conformations in dilute aqueous solutions, we demonstrate, using UV Raman spectroscopy, that they become predominately alpha-helical in solution at pH > 10, in high ionic strength solutions, and in the presence of trifluoroethanol (TFE) and dodecylphosphocholine (DPC) micelles. In this paper, we examine the effects of electrostatic and hydrophobic interactions that control folding of BI and BIII by systematically monitoring their secondary structures as a function of solution conditions. We determine the BI and BIII secondary structure contents by using the quantitative UV Raman methodology of Chi et al. (1998. Biochemistry. 37:2854-2864). Our findings suggest that the alpha-helix turn in BIII at neutral pH is stabilized by a salt bridge between residues Asp2 and Lys5. This initial alpha-helical turn results in different BI and BIII alpha-helical folding mechanisms observed in high pH and high salt concentrations: BIII folds from its single alpha-helix turn close to its N-terminal, whereas the BI alpha-helix probably nucleates within the C-terminal half. We also used quasielastic light scattering to demonstrate that the BI and BIII alpha-helix formation in 0.2 M Ca(ClO4)2 is accompanied by formation of trimers and hexamers, respectively.     

The structural basis for molecular recognition by the vitamin B 12 RNA aptamer

Previous solution structures of ligand-binding RNA aptamers have shown that molecular recognition is achieved by the folding of an initially unstructured RNA around its cognate ligand, coupling the processes of RNA folding and binding. The 3 A crystal structure of the cyanocobalamin (vitamin B12) aptamer reported here suggests a different approach to molecular recognition in which elements of RNA secondary structure combine to create a solvent-accessible docking surface for a large, complex ligand. Central to this structure is a locally folding RNA triplex, stabilized by a novel three-stranded zipper. Perpendicular stacking of a duplex on this triplex creates a cleft that functions as the vitamin B12 binding site. Complementary packing of hydrophobic surfaces, direct hydrogen bonding and dipolar interactions between the ligand and the RNA appear to contribute to binding. The nature of the interactions that stabilize complex formation and the possible uncoupling of folding and binding for this RNA suggest a strong mechanistic similarity to typical protein-ligand complexes.     

Folding propensities of peptide fragments of myoglobin.

Myoglobin has been studied extensively as a paradigm for protein folding. As part of an ongoing study of potential folding initiation sites in myoglobin, we have synthetized a series of peptides covering the entire sequence of sperm whale myoglobin. We report here on the conformation preferences of a series of peptides that cover the region from the A helix to the FG turn. Structural propensities were determined using circular dichroism and nuclear magnetic resonance spectroscopy in aqueous solution, trifluoroethanol, and methanol. Peptides corresponding to helical regions in the native protein, namely the B, C, D, and E helices, populate the alpha region of (phi, psi) space in water solution but show no measurable helix formation except in the presence of trifluoroethanol. The F-helix sequence has a much lower propensity to populate helical conformations even in TFE. Despite several attempts, we were not successful in synthesizing a peptide corresponding to the A-helix region that was soluble in water. A peptide termed the AB domain was constructed spanning the A- and B-helix sequences. The AB domain is not soluble in water, but shows extensive helix formation throughout the peptide when dissolved in methanol, with a break in the helix at a site close to the A-B helix junction in the intact folded myoglobin protein. With the exception of one local preference for a turn conformation stabilized by hydrophobic interactions, the peptides corresponding to turns in the folded protein do not measurably populate beta-turn conformations in water, and the addition of trifluoroethanol does not enhance the formation of either helical or turn structure. In contrast to the series of peptides described here, either studies of peptides from the GH region of myoglobin show a marked tendency to populate helical structures (H), nascent helical structures (G), or turn conformations (GH peptide) in water solution. This region, together with the A-helix and part of the B-helix, has been shown to participate in an early folding intermediate. The complete analysis of conformational properties of isolated myoglobin peptides supports the hypothesis that spontaneous secondary structure formation in local regions of the polypeptide may play an important role in the initiation of protein folding.     

Importance of secondary structural specificity determinants in protein folding: insertion of a native beta-sheet sequence into an alpha-helical coiled-coil

To examine how a short secondary structural element derived from a native protein folds when in a different protein environment, we inserted an 11-residue beta-sheet segment (cassette) from human immunoglobulin fold, Fab new, into an alpha-helical coiled-coil host protein (cassette holder). This de novo design protein model, the structural cassette mutagenesis (SCM) model, allows us to study protein folding principles involving both short- and long-range interactions that affect secondary structure stability and conformation. In this study, we address whether the insertion of this beta-sheet cassette into the alpha-helical coiled-coil protein would result in conformational change nucleated by the long-range tertiary stabilization of the coiled-coil, therefore overriding the local propensity of the cassette to form beta-sheet, observed in its native immunoglobulin fold. The results showed that not only did the nucleating helices of the coiled-coil on either end of the cassette fail to nucleate the beta-sheet cassette to fold with an alpha-helical conformation, but also the entire chimeric protein became a random coil. We identified two determinants in this cassette that prevented coiled-coil formation: (1) a tandem dipeptide NN motif at the N-terminal of the beta-sheet cassette, and (2) the hydrophilic Ser residue, which would be buried in the hydrophobic core if the coiled-coil structure were to fold. By amino acid substitution of these helix disruptive residues, that is, either the replacement of the NN motif with high helical propensity Ala residues or the substitution of Ser with Leu to enhance hydrophobicity, we were able to convert the random coil chimeric protein into a fully folded alpha-helical coiled-coil. We hypothesized that this NN motif is a "secondary structural specificity determinant" which is very selective for one type of secondary structure and may prevent neighboring residues from adopting an alternate protein fold. These sequences with secondary structural specificity determinants have very strong local propensity to fold into a specific secondary structure and may affect overall protein folding by acting as a folding initiation site.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Solvent accessibility, protein surfaces, and protein folding.

Studies of the native structures of proteins, together with measurements of the thermodynamic properties of the transition between unfolded and native states, have defined the major components of the forces that stabilize native protein structures. However, the nature of the intermediates in the folding process remains largely hypothetical. It is a fairly widespread and not implausible assumption that the intermediates in the folding of a monomeric protein contain the same kinds of secondary and tertiary structures that appear in the native conformation, and that, although unstable, their lifetimes are prolonged by forces similar to those that stabilize the native structure. We wished to examine what happens if, during the folding of a monomeric protein, regions of secondary structure come together to form an intermediate of reduced instability. We applied calculations of accessible surface area (a measure of hydrophobic stabilization) and parameterized nonbonded energy calculations (measuring the strengths of van der Waals forces) to identify the kinds of stabilizing interactions that might be available to such an intermediate. First, we analyzed the total buried surface area of two types of proteins into contributions from formation of secondary structure alone, interaction of pairs of secondary-structural elements, the formation of the structure alone, interaction of pairs of secondary-structural elements, the formation of the complete secondary structure without the turns, and the complete native structure. The formation of secondary structure alone, without tertiary-structural interactions, buries roughly half the surface that the complete structure does. We then analyzed in more detail the approach of two alpha-helices to form a complex, as an illustrative example of the nature of the interaction between compact structural units which remain fairly rigid during their interaction. Many features of the results are not limited to the interaction of alpha-helices. (The results therefore neither confirm nor refute the hypothesis that alpha-helices are intermediates in the folding proteins). We find that the first forces to be felt upon approach arise from solvent conditions on the relative position and orientation of the two helices as does the close packing which optimizes the van der Waals interactions at shorter distances apart. Therefore there appears to be a range of distances in which hydrophobic interactions could create a nonspecific complex between two helices in which the side chains might have sufficient time to seek the proper interdigitation observed in the native structure, where the two helices are in intimate contact. Indeed, we find that only in the final stages of approach is the native geometry the most stable; in the region in which solvent-exclusion forces predominate, the conformation with helix axes parallel is more stable than the native conformation, in the cases we examined...     

Conformational and dynamic characterization of the molten globule state of an apomyoglobin mutant with an altered folding pathway.

Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.