De novo design of the hydrophobic core of ubiquitin.

We have previously reported the development and evaluation of a computational program to assist in the design of hydrophobic cores of proteins. In an effort to investigate the role of core packing in protein structure, we have used this program, referred to as Repacking of Cores (ROC), to design several variants of the protein ubiquitin. Nine ubiquitin variants containing from three to eight hydrophobic core mutations were constructed, purified, and characterized in terms of their stability and their ability to adopt a uniquely folded native-like conformation. In general, designed ubiquitin variants are more stable than control variants in which the hydrophobic core was chosen randomly. However, in contrast to previous results with 434 cro, all designs are destabilized relative to the wild-type (WT) protein. This raises the possibility that beta-sheet structures have more stringent packing requirements than alpha-helical proteins. A more striking observation is that all variants, including random controls, adopt fairly well-defined conformations, regardless of their stability. This result supports conclusions from the cro studies that non-core residues contribute significantly to the conformational uniqueness of these proteins while core packing largely affects protein stability and has less impact on the nature or uniqueness of the fold. Concurrent with the above work, we used stability data on the nine ubiquitin variants to evaluate and improve the predictive ability of our core packing algorithm. Additional versions of the program were generated that differ in potential function parameters and sampling of side chain conformers. Reasonable correlations between experimental and predicted stabilities suggest the program will be useful in future studies to design variants with stabilities closer to that of the native protein. Taken together, the present study provides further clarification of the role of specific packing interactions in protein structure and stability, and demonstrates the benefit of using systematic computational methods to predict core packing arrangements for the design of proteins.     

Side chain burial and hydrophobic core packing in protein folding transition states

A critical step in the folding pathway of globular proteins is the formation of a tightly packed hydrophobic core. Several mutational studies have addressed the question of whether tight packing interactions are present during the rate-limiting step of folding. In some of these investigations, substituted side chains have been assumed to form native-like interactions in the transition state when the folding rates of mutant proteins correlate with their native-state stabilities. Alternatively, it has been argued that side chains participate in nonspecific hydrophobic collapse when the folding rates of mutant proteins correlate with side-chain hydrophobicity. In a reanalysis of published data, we have found that folding rates often correlate similarly well, or poorly, with both native-state stability and side-chain hydrophobicity, and it is therefore not possible to select an appropriate transition state model based on these one-parameter correlations. We show that this ambiguity can be resolved using a two-parameter model in which side chain burial and the formation of all other native-like interactions can occur asynchronously. Notably, the model agrees well with experimental data, even for positions where the one-parameter correlations are poor. We find that many side chains experience a previously unrecognized type of transition state environment in which specific, native-like interactions are formed, but hydrophobic burial dominates. Implications of these results to the design and analysis of protein folding studies are discussed.     

Charge centers and formation of the protein folding core

Electrostatic interactions are important for protein folding. At low resolution, the electrostatic field of the whole molecule can be described in terms of charge center(s). To study electrostatic effects, the centers of positive and negative charge were calculated for 20 small proteins of known structure, for which hydrogen exchange cores had been determined experimentally. Two observations seem to be important. First, in all 20 proteins studied 30-100% of the residues forming hydrogen exchange core(s) were clustered around the charge centers. Moreover, in each protein more than half of the core sequences are located near the centers of charge. Second, the general architecture of all-alpha proteins from the set seems to be stabilized by interactions of residues surrounding the charge centers. In most of the alpha-beta proteins, either or both of the centers are located near a pair of consecutive strands, and this is even more characteristic for alpha/Beta and all-beta structures. Consecutive strands are very probable sites of early folding events. These two points lead to the conclusion that charge centers, defined solely from the structure of the folded protein may indicate the location of a protein's hydrogen exchange/folding core. In addition, almost all the proteins contain well-conserved continuous hydrophobic sequences of three or more residues located in the vicinity of the charge centers. These hydrophobic sequences may be primary nucleation sites for protein folding. The results suggest the following scheme for the order of events in folding: local hydrophobic nucleation, electrostatic collapse of the core, global hydrophobic collapse, and slow annealing to the native state. This analysis emphasizes the importance of treating electrostatics during protein-folding simulations.     

Intermediates and the folding of proteins L and G

We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted beta-1 and beta-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third beta-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment.     

Modularity of the hydrophobic core and evolution of functional diversity in fold A glycosyltransferases

Hydrophobic cores are fundamental structural properties of proteins typically associated with protein folding and stability; however, how the hydrophobic core shapes protein evolution and function is poorly understood. Here, we investigated the role of conserved hydrophobic cores in fold-A glycosyltransferases (GT-As), a large superfamily of enzymes that catalyze formation of glycosidic linkages between diverse donor and acceptor substrates through distinct catalytic mechanisms (inverting versus retaining). Using hidden Markov models and protein structural alignments, we identify similarities in the phosphate-binding cassette (PBC) of GT-As and unrelated nucleotide-binding proteins, such as UDP-sugar pyrophosphorylases. We demonstrate that GT-As have diverged from other nucleotide-binding proteins through structural elaboration of the PBC and its unique hydrophobic tethering to the F-helix, which harbors the catalytic base (xED-Asp). While the hydrophobic tethering is conserved across diverse GT-A fold enzymes, some families, such as B3GNT2, display variations in tethering interactions and core packing. We evaluated the structural and functional impact of these core variations through experimental mutational analysis and molecular dynamics simulations and find that some of the core mutations (T336I in B3GNT2) increase catalytic efficiency by modulating the conformational occupancy of the catalytic base between “D-in” and acceptor-accessible “D-out” conformation. Taken together, our studies support a model of evolution in which the GT-A core evolved progressively through elaboration upon an ancient PBC found in diverse nucleotide-binding proteins, and malleability of this core provided the structural framework for evolving new catalytic and substrate-binding functions in extant GT-A fold enzymes.     

Monitoring protein conformation along the pathway of chaperonin-assisted folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.     

Computational protein design with explicit consideration of surface hydrophobic patches

De novo protein design requires the identification of amino-acid sequences that favor the target-folded conformation and are soluble in water. One strategy for promoting solubility is to disallow hydrophobic residues on the protein surface during design. However, naturally occurring proteins often have hydrophobic amino acids on their surface that contribute to protein stability via the partial burial of hydrophobic surface area or play a key role in the formation of protein-protein interactions. A less restrictive approach for surface design that is used by the modeling program Rosetta is to parameterize the energy function so that the number of hydrophobic amino acids designed on the protein surface is similar to what is observed in naturally occurring monomeric proteins. Previous studies with Rosetta have shown that this limits surface hydrophobics to the naturally occurring frequency (～28%), but that it does not prevent the formation of hydrophobic patches that are considerably larger than those observed in naturally occurring proteins. Here, we describe a new score term that explicitly detects and penalizes the formation of hydrophobic patches during computational protein design. With the new term, we are able to design protein surfaces that include hydrophobic amino acids at naturally occurring frequencies, but do not have large hydrophobic patches. By adjusting the strength of the new score term, the emphasis of surface redesigns can be switched between maintaining solubility and maximizing folding free energy.     

Novel approach for alpha-helical topology prediction in globular proteins: generation of interhelical restraints

The protein folding problem represents one of the most challenging problems in computational biology. Distance constraints and topology predictions can be highly useful for the folding problem in reducing the conformational space that must be searched by deterministic algorithms to find a protein structure of minimum conformational energy. We present a novel optimization framework for predicting topological contacts and generating interhelical distance restraints between hydrophobic residues in alpha-helical globular proteins. It should be emphasized that since the model does not make assumptions about the form of the helices, it is applicable to all alpha-helical proteins, including helices with kinks and irregular helices. This model aims at enhancing the ASTRO-FOLD protein folding approach of Klepeis and Floudas (Journal of Computational Chemistry 2003;24:191-208), which finds the structure of global minimum conformational energy via a constrained nonlinear optimization problem. The proposed topology prediction model was evaluated on 26 alpha-helical proteins ranging from 2 to 8 helices and 35 to 159 residues, and the best identified average interhelical distances corresponding to the predicted contacts fell below 11 A in all 26 of these systems. Given the positive results of applying the model to several protein systems, the importance of interhelical hydrophobic-to-hydrophobic contacts in determining the folding of alpha-helical globular proteins is highlighted.     

Im7 folding mechanism: misfolding on a path to the native state

Many proteins populate collapsed intermediate states during folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical mechanism of protein folding and explain why the misfolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in folding, even for small proteins with simple topologies.     

Surface hydrophobic groups, stability, and flip-flopping in lattice proteins

Two-dimensional lattice protein models were studied in two approximations of the conformational equilibrium to elucidate the role of surface hydrophobic groups in their stabilities. We demonstrate that stability of any compactly folded sequence is determined by its ability to "flip-flop" (refold) into alternative compact structures. The degree of stability required for folded sequences determines the average numbers of surface hydrophobic groups in stable lattice structures which are in good agreement with ratios of core to surface hydrophobic groups in real proteins. However, the average destabilization of the native structure per surface hydrophobic group is small (0-0.25 kcal/mol), often disagrees with the free energies derived from the ratios of core to surface hydrophobic groups in the same structures, and has a combinatorial entropic nature independent of the strength of structure stabilizing interactions. This suggests that the free energies derived from the core to surface ratios of hydrophobic groups in real proteins have little to do with folding thermodynamics. On average, sequences with highly stable native structures are the least hydrophobic. The results suggest that in designing novel stable proteins hydrophobic groups on the surface should be avoided to reduce the possibility of flip-flopping. The average stability of highly designable structures is never higher than that of some low designability structures, contrary to the accepted view. In the equilibrium approximation with alternative compact and partially unfolded structures, the requirement of high stability selects a unique 5 x 5 structure formed by only a few sequences, suggesting much stronger sequence selectivity than commonly thought.     

An empirical energy function for threading protein sequence through the folding motif

In this paper we present a new residue contact potantial derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by “threading” different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161-protcin data set. They also identify correct models with the core folding motifs of heme-rythrin and immunoglobulin McPC603 V₁-do- main, among millions of alternatives possible when we align subsequences with α-helices and β-strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that “threading” may be useful for structure prediction by recognition of folding motif. © 1993 Wiley-Liss, Inc.     

Robustness of protein folding kinetics to surface hydrophobic substitutions

We use both combinatorial and site-directed mutagenesis to explore the consequences of surface hydrophobic substitutions for the folding of two small single domain proteins, the src SH3 domain, and the IgG binding domain of Peptostreptococcal protein L. We find that in almost every case, destabilizing surface hydrophobic substitutions have much larger effects on the rate of unfolding than on the rate of folding, suggesting that nonnative hydrophobic interactions do not significantly interfere with the rate of core assembly.     

The nature of the free energy barriers to two-state folding

We introduce a simple procedure to analyze the temperature dependence of the folding and unfolding rates of two-state proteins. We start from the simple transition-state-like rate expression: k = D(eff)exp(-DeltaG(TS)/RT), in which upper and lower bounds for the intra-chain effective diffusion coefficient (D(eff)) are obtained empirically using the timescales of elementary processes in protein folding. From the changes in DeltaG(TS) as a function of temperature, we calculate enthalpies and heat capacities of activation, together with the more elusive entropies of activation. We then estimate the conformational entropy of the transition state by extrapolation to the temperature at which the solvation entropy vanishes by cancellation between polar and apolar terms. This approach is based on the convergence temperatures for the entropy of solvating apolar (approximately 385 K) and polar groups (approximately 335 K), the assumption that the structural properties of the transition state are somewhere in between the unfolded and folded states, and the established relationship between observed heat capacity and solvent accessibility.1 To circumvent the lack of structural information about transition states, we use the empirically determined heat capacities of activation as constraints to identify the extreme values of the transition state conformational entropy that are consistent with experiment. The application of this simple approach to six two-state folding proteins for which there is temperature-dependent data available in the literature provides important clues about protein folding. For these six proteins, we obtain an average equilibrium cost in conformational entropy of -4.3 cal x mol(-1)K(-1)per residue, which is in close agreement to previous empirical and computational estimates of the same quantity. Furthermore, we find that all these proteins have a conformationally diverse transition state, with more than half of the conformational entropy of the unfolded state. In agreement with predictions from theory and computer simulations, the transition state signals the change from a regime dominated by loss in conformational entropy to one driven by the gain in stabilization free energy (i.e., including protein interactions and solvation effects). Moreover, the height of the barrier is determined by how much stabilization free energy is realized at that point, which is related to the relative contribution of local versus non-local interactions. A remarkable observation is that the fraction of conformational entropy per residue that is present in the transition state is very similar for the six proteins in this study. Based on this commonality, we propose that the observed change in thermodynamic regime is connected to a change in the pattern of structure formation: from one driven by formation of pairwise interactions to one dominated by coupling of the networks of interactions involved in forming the protein core. In this framework, the barrier to two-state folding is crossed when the folding protein reaches a "critical native density" that allows expulsion of remaining interstitial water and consolidation of the core. The principle of critical native density should be general for all two-state proteins, but can accommodate different folding mechanisms depending on the particularities of the structure and sequence.     

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.     

Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding

Development of a tightly packed hydrophobic core drives the folding of water-soluble globular proteins and is a key determinant of protein stability. Despite this, there remains much to be learnt about how and when the hydrophobic core becomes desolvated and tightly packed during protein folding. We have used the bacterial immunity protein Im7 to examine the specificity of hydrophobic core packing during folding. This small, four-helix protein has previously been shown to fold via a compact three-helical intermediate state. Here, overpacking substitutions, in which residue side-chain size is increased, were used to examine the specificity and malleability of core packing in the folding intermediate and rate-limiting transition state. In parallel, polar groups were introduced into the Im7 hydrophobic core via Val→Thr or Phe→Tyr substitutions and used to determine the solvation status of core residues at different stages of folding. Over 30 Im7 variants were created allowing both series of substitutions to cover all regions of the protein structure. Φ-value analysis demonstrated that the major changes in Im7 core solvation occur prior to the population of the folding intermediate, with key regions involved in docking of the short helix III remaining solvent-exposed until after the rate-limiting transition state has been traversed. In contrast, overpacking core residues revealed that some regions of the native Im7 core are remarkably malleable to increases in side-chain volume. Overpacking residues in other regions of the Im7 core result in substantial (> 2.5 kJ mol^− 1) destabilisation of the native structure or even prevents efficient folding to the native state. This study provides new insights into Im7 folding; demonstrating that whilst desolvation occurs early during folding, adoption of a specifically packed core is achieved only at the very last step in the folding mechanism.     

Studies of protein-protein interfaces: a statistical analysis of the hydrophobic effect.

Data sets of 362 structurally nonredundant protein-protein interfaces and of 57 symmetry-related oligomeric interfaces have been used to explore whether the hydrophobic effect that guides protein folding is also the main driving force for protein-protein associations. The buried nonpolar surface area has been used to measure the hydrophobic effect. Our analysis indicates that, although the hydrophobic effect plays a dominant role in protein-protein binding, it is not as strong as that observed in the interior of protein monomers. Comparison of interiors of the monomers with those of the interfaces reveals that, in general, the hydrophobic amino acids are more frequent in the interior of the monomers than in the interior of the protein-protein interfaces. On the other hand, a higher proportion of charged and polar residues are buried at the interfaces, suggesting that hydrogen bonds and ion pairs contribute more to the stability of protein binding than to that of protein folding. Moreover, comparison of the interior of the interfaces to protein surfaces indicates that the interfaces are poorer in polar/charged than the surfaces and are richer in hydrophobic residues. The interior of the interfaces appears to constitute a compromise between the stabilization contributed by the hydrophobic effect on the one hand and avoiding patches on the protein surfaces that are too hydrophobic on the other. Such patches would be unfavorable for the unassociated monomers in solution. We conclude that, although the types of interactions are similar between protein-protein interfaces and single-chain proteins overall, the contribution of the hydrophobic effect to protein-protein associations is not as strong as to protein folding. This implies that packing patterns and interatom, or interresidue, pairwise potential functions, derived from monomers, are not ideally suited to predicting and assessing ligand associations or design. These would perform adequately only in cases where the hydrophobic effect at the binding site is substantial.     

The role of aromatic residues in the hydrophobic core of the villin headpiece subdomain

Small autonomously folding proteins are of interest as model systems to study protein folding, as the same molecule can be used for both experimental and computational approaches. The question remains as to how well these minimized peptide model systems represent larger native proteins. For example, is the core of a minimized protein tolerant to mutation like larger proteins are? Also, do minimized proteins use special strategies for specifying and stabilizing their folded structure? Here we examine these questions in the 35‐residue autonomously folding villin headpiece subdomain (VHP subdomain). Specifically, we focus on a cluster of three conserved phenylalanine (F) residues F47, F51, and F58, that form most of the hydrophobic core. These three residues are oriented such that they may provide stabilizing aromatic–aromatic interactions that could be critical for specifying the fold. Circular dichroism and 1D‐NMR spectroscopy show that point mutations that individually replace any of these three residues with leucine were destabilized, but retained the native VHP subdomain fold. In pair‐wise replacements, the double mutant that retains F58 can adopt the native fold, while the two double mutants that lack F58 cannot. The folding of the double mutant that retains F58 demonstrates that aromatic–aromatic interactions within the aromatic cluster are not essential for specifying the VHP subdomain fold. The ability of the VHP subdomain to tolerate mutations within its hydrophobic core indicates that the information specifying the three dimensional structure is distributed throughout the sequence, as observed in larger proteins. Thus, the VHP subdomain is a legitimate model for larger, native proteins.     

Increased sequence hydrophobicity reduces conformational specificity: A mutational case study of the Arc repressor protein

The amino-acid sequences of soluble, globular proteins must have hydrophobic residues to form a stable core, but excess sequence hydrophobicity can lead to loss of native state conformational specificity and aggregation. Previous studies of polar-to-hydrophobic mutations in the β-sheet of the Arc repressor dimer showed that a single substitution at position 11 (N11L) leads to population of an alternate dimeric fold in which the β-sheet is replaced by helix. Two additional hydrophobic mutations at positions 9 and 13 (Q9V and R13V) lead to population of a differently folded octamer along with both dimeric folds. Here we conduct a comprehensive study of the sequence determinants of this progressive loss of fold specificity. We find that the alternate dimer-fold specifically results from the N11L substitution and is not promoted by other hydrophobic substitutions in the β-sheet. We also find that three highly hydrophobic substitutions at positions 9, 11, and 13 are necessary and sufficient for oligomer formation, but the oligomer size depends on the identity of the hydrophobic residue in question. The hydrophobic substitutions increase thermal stability, illustrating how increased hydrophobicity can increase folding stability even as it degrades conformational specificity. The oligomeric variants are predicted to be aggregation-prone but may be hindered from doing so by proline residues that flank the β-sheet region. Loss of conformational specificity due to increased hydrophobicity can manifest itself at any level of structure, depending upon the specific mutations and the context in which they occur.     

Structured disorder and conformational selection

Traditionally, molecular disorder has been viewed as local or global instability. Molecules or regions displaying disorder have been considered inherently unstructured. The term has been routinely applied to cases for which no atomic coordinates can be derived from crystallized molecules. Yet, even when it appears that the molecules are disordered, prevailing conformations exist, with population times higher than those of all alternate conformations. Disordered molecules are the outcome of rugged energy landscapes away from the native state around the bottom of the funnel. Ruggedness has a biological function, creating a distribution of structured conformers that bind via conformational selection, driving association and multimolecular complex formation, whether chain-linked in folding or unlinked in binding. We classify disordered molecules into two types. The first type possesses a hydrophobic core. Here, even if the native conformation is unstable, it still has a large enough population time, enabling its experimental detection. In the second type, no such hydrophobic core exists. Hence, the native conformations of molecules belonging to this category have shorter population times, hindering their experimental detection. Although there is a continuum of distribution of hydrophobic cores in proteins, an empirical, statistically based hydrophobicity function may be used as a guideline for distinguishing the two disordered molecule types. Furthermore, the two types relate to steps in the protein folding reaction. With respect to protein design, this leads us to propose that engineering-optimized specific electrostatic interactions to avoid electrostatic repulsion would reduce the type I disordered state, driving the molten globule (MG) --> native (N) state. In contrast, for overcoming the type II disordered state, in addition to specific interactions, a stronger hydrophobic core is also indicated, leading to the denatured --> MG --> N state.