Simulations of reversible protein aggregate and crystal structure.

We simulated the structure of reversible protein aggregates as a function of protein surface characteristics, protein-protein interaction energies, and the entropic penalty accompanying the immobilization of protein in a solid phase. These simulations represent an extension of our previous work on kinetically irreversible protein aggregate structure and are based on an explicit accounting of the specific protein-protein interactions that occur within reversible aggregates and crystals. We considered protein monomers with a mixture of hydrophobic and hydrophilic surface regions suspended in a polar solvent; the energetic driving force for aggregation is provided by the burial of solvent-exposed hydrophobic surface area. We analyzed the physical properties of the generated aggregates, including density, protein-protein contact distributions, solvent accessible surface area, porosity, and order, and compared our results with the protein crystallization literature as well as with the kinetically irreversible case. The physical properties of reversible aggregates were consonant with those observed for the irreversible aggregates, although in general, reversible aggregates were more stable energetically and were more crystal-like in their order content than their irreversible counterparts. The reversible aggregates were less dense than the irreversible aggregates, indicating that the increased energetic stability is derived primarily from the optimality rather than the density of the packing in the solid phase. The extent of hydrophobic protein-protein contacts and solvent-exposed surface area within the aggregate phase depended on the aggregation pathway: reversible aggregates tended to have a greater proportion of hydrophobic-hydrophobic contacts and a smaller fraction of hydrophobic solvent-exposed surface area. Furthermore, the arrangement of hydrophobic patches on the protein surface played a major role in the distribution of protein contacts and solvent content. This was readily reflected in the order of the aggregates: the greater the contiguity of the hydrophobic patches on the monomer surface, the less ordered the aggregates became, despite the opportunities for rearrangement offered by a reversible pathway. These simulations have enhanced our understanding of the impact of protein structural motifs on aggregate properties and on the demarcation between aggregation and crystallization.     

Packing forces in nine crystal forms of cutinase

During the characterization of mutants and covalently inhibited complexes of Fusarium solani cutinase, nine different crystal forms have been obtained so far. Protein mutants with a different surface charge distribution form new intermolecular salt bridges or long-range electrostatic interactions that are accompanied by a change in the crystal packing. The whole protein surface is involved in the packing contacts and the hydrophobicities of the protein surfaces in mutual contact turned out to be noncorrelated, which indicates that the packing interactions are nonspecific. In the case of the hydrophobic variants, the packing contacts showed some specificity, as the protein in the crystal tends to form either crystallographic or noncrystallographic dimers, which shield the hydrophobic surface from the solvent. The likelihood of surface atoms to be involved in a crystal contact is the same for both polar and nonpolar atoms. However, when taking areas in the 200–600 Ų range, instead of individual atoms, the either highly hydrophobic or highly polar surface regions were found to have an increased probability of establishing crystal lattice contacts. The protein surface surrounding the active-site crevice of cutinase constitutes a large hydrophobic area that is involved in packing contacts in all the various crystalline contexts. Proteins 31:320–333, 1998. © 1998 Wiley-Liss, Inc.     

Excluded volume approximation to protein-solvent interaction. The solvent contact model.

Important properties of globular proteins, such as the stability of its folded state, depend sensitively on interactions with solvent molecules. Existing methods for estimating these interactions, such as the geometrical surface model, are either physically misleading or too time consuming to be applied routinely in energy calculations. As an alternative, we derive here a simple model for the interactions between protein atoms and solvent atoms in the first hydration layer, the solvent contact model, based on the conservation of the total number of atomic contacts, a consequence of the excluded-volume effect. The model has the conceptual advantage that protein-protein contacts and protein-solvent contacts are treated in the same language and the technical advantage that the solvent term becomes a particularly simple function of interatomic distances. The model allows rapid calculation of any physical property that depends only on the number and type of protein-solvent nearest-neighbor contacts. We propose use of the method in the calculation of protein solvation energies, conformational energy calculations, and molecular dynamics simulations.     

Structural mapping of an aggregation nucleation site in a molten globule intermediate.

Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation.     

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)     

Refolding of chemically denatured alpha-amylase in dilution additive mode

Cyclodextrins (CDs) possess hydrophobic surfaces, which probably shield the hydrophobic surfaces of denatured proteins and prevent the direct interactions between the surfaces which are believed to be responsible for protein aggregation during refolding process. This probability was evaluated by studying the refolding process of denatured alpha-amylase in the presence and absence of alpha-CD, as a dilution additive agent. Our data indicate that in the presence of 100 mM alpha-CD in the refolding buffer, the extent of aggregation reduces by almost 90%. Spectrofluorometric analysis of the refolding intermediate(s) also indicates that the tertiary structure of the refolded alpha-amylase, in the presence of alpha-CD, is very similar to the tertiary structure of the native protein. However, this similarity was distorted upon addition of exogenous hydrophobic (aliphatic or aromatic) amino acids to the refolding buffer, meaning that the hydrophobic interactions between alpha-CD and the denatured protein play significant role in preventing aggregate formation. In addition, by weakening the extent of these hydrophobic interactions by adding polarity-reducing agent (e.g. ethylene glycol) to the refolding buffer, more aggregates were formed. In contrast, strengthening these interactions by enhancing the ionic strength of the refolding buffer made these hydrophobic interactions very strong. Therefore, alpha-CD could not depart from the protein/alpha-CD complex, as it usually does during the process of refolding. As a result, more aggregates were formed in the presence of alpha-CD compared to the corresponding control samples.     

Anti-cooperativity and cooperativity in hydrophobic interactions: Three-body free energy landscapes and comparison with implicit-solvent potential functions for proteins

Potentials of mean force (PMFs) of three-body hydrophobic association are investigated to gain insight into similar processes in protein folding. Free energy landscapes obtained from explicit simulations of three methanes in water are compared with that predicted by popular implicit-solvent effective potentials for the study of proteins. Explicit-water simulations show that for an extended range of three-methane configurations, hydrophobic association at 25 degrees C under atmospheric pressure is mostly anti-cooperative, that is, less favorable than if the interaction free energies were pairwise additive. Effects of free energy nonadditivity on the kinetic path of association and the temperature dependence of additivity are explored by using a three-methane system and simplified chain models. The prevalence of anti-cooperativity under ambient conditions suggests that driving forces other than hydrophobicity also play critical roles in protein thermodynamic cooperativity. We evaluate the effectiveness of several implicit-solvent potentials in mimicking explicit water simulated three-body PMFs. The favorability of the contact free energy minimum is found to be drastically overestimated by solvent accessible surface area (SASA). Both the SASA and a volume-based Gaussian solvent exclusion model fail to predict the desolvation barrier. However, this barrier is qualitatively captured by the molecular surface area model and a recent "hydrophobic force field." None of the implicit-solvent models tested are accurate for the entire range of three-methane configurations and several other thermodynamic signatures considered.     

Stability and structure of oligomers of the Alzheimer peptide Abeta16-22: from the dimer to the 32-mer

Several neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases are associated with amyloid fibrils formed by different polypeptides. We probe the structure and stability of oligomers of different sizes of the fragment Abeta(16-22) of the Alzheimer beta-amyloid peptide using atomic-detail molecular dynamics simulations with explicit solvent. We find that only large oligomers form a stable beta-sheet aggregate, the minimum nucleus size being of the order of 8-16 peptides. This effect is attributed to better hydrophobic contacts and a better shielding of backbone-backbone hydrogen bonds from the solvent in bigger assemblies. Moreover, the observed stability of beta-sheet aggregates with a different number of layers can be explained on the basis of their solvent-accessible surface area. Depending on the stacking interface between the sheets, we observe straight or twisted structures, which could be linked to the experimentally observed polymorphism of amyloid fibrils. To compare our 32-mer structure to experimental data, we calculate its x-ray diffraction pattern. Good agreement is found between experimentally and theoretically determined reflections, suggesting that our model indeed closely resembles the structures found in vitro.     

Molecular mechanisms of protein-protein recognition: whether the surface placed charged residues determine the recognition process?

We studied the structure and composition of contact areas in 812 different kind dimeric protein-protein complexes from Brookhaven data base (PDB ) in order to reveal their pecularities with regard to protein-protein recognition. We have found, that the large portion of complexes (approximately 70%) have oppositely charged residues in the contact areas (interfaces) on the subunits surfaces, which form electrostatic contacts - R:E, R:D, K:E, K:D, H:E, H:D. These results are consistent with the current view that high rate complex formation may be driven by the long-range electrostatic interaction between charged AA residues of subunits surfaces. However, there are many complexes among the studied ones (approximately 30%), which have no electrostatic contacts at all in their contact area. Thus a question arises: what forces account for high complex formation rates (i.e. for the distant orienting of subunits before encounter) by forming complexes where the surface contact areas lack electrostatic contacts? We believe that the long-range orienting electrostatic interaction of subunits may account for all cases of efficient complex formation if one drops the traditional view that protein subunits interact mainly through their surfaces. We suggest that the distant orienting being due to the electrostatic interaction between the whole aggregates of partial electric charges of atoms of each complex subunits. Our preliminary model calculations (unpublished) made for ribonuclease dimer (does not have electrostatic contacts) conform this suggestion.     

Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Towards accurate residue-residue hydrophobic contact prediction for alpha helical proteins via integer linear optimization

A new optimization-based method is presented to predict the hydrophobic residue contacts in alpha-helical proteins. The proposed approach uses a high resolution distance dependent force field to calculate the interaction energy between different residues of a protein. The formulation predicts the hydrophobic contacts by minimizing the sum of these contact energies. These residue contacts are highly useful in narrowing down the conformational space searched by protein structure prediction algorithms. The proposed algorithm also offers the algorithmic advantage of producing a rank ordered list of the best contact sets. This model was tested on four independent alpha-helical protein test sets and was found to perform very well. The average accuracy of the predictions (separated by at least six residues) obtained using the presented method was approximately 66% for single domain proteins. The average true positive and false positive distances were also calculated for each protein test set and they are 8.87 and 14.67 A, respectively.     

Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1

In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine-betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQ aggregates.     

Transient aggregation and stable dimerization induced by introducing an Alzheimer sequence into a water-soluble protein

Transient contacts between denatured polypeptide chains are likely to play an important part in the initial stages of protein aggregation and fibrillation. To analyze the nature of such contacts, we have carried out a protein engineering study of the 102-residue protein U1A, which aggregates transiently in the wild-type form during refolding from the guanidinium chloride-denatured state. We have prepared a series of mutants with increased aggregation tendencies by increasing the homology between two beta-strands of U1A and the Alzheimer peptide (beta-AP). These mutants undergo transient aggregation during refolding, as measured by concentration dependence, double-jump experiments, and binding of ANS, a probe for exposed hydrophobic patches on protein surfaces. The propensity to aggregate increases with increasing homology to beta-AP. Further, the degree of transient ANS binding correlates reasonably well with the structural parameters recently shown to play a role in the fibrillation of natively unfolded proteins. Two mutants highly prone to transient aggregation, U1A-J and U1A-G, were also studied by NMR. Secondary structural elements of the U1A-J construct (with lower beta-AP homology) are very similar to those observed in U1A-wt. In contrast, the high-homology construct U1A-G exhibits local unfolding of the C-terminal helix, which packs against the beta-sheet in the wild-type protein. U1A-G is mainly dimeric according to (15)N spin relaxation data, and the dimer interface most likely involves the beta-sheet. Our data suggest that the transient aggregate relies on specific intermolecular interactions mediated by structurally flexible regions and that contacts may be formed in different beta-strand registers.     

Structural Reorganization of α-Synuclein at Low pH Observed by NMR and REMD Simulations

α-Synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's disease. The conversion from monomer to aggregate is complex, and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and replica exchange molecular dynamics simulations indicate that there is a significant structural reorganization within the low-pH ensemble relative to that at neutral pH in terms of long-range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH, there is a very heterogeneous ensemble with transient contacts between the N-terminus and the non-amyloid β component (NAC); however, at low pH, there is a more homogeneous ensemble that exhibits strong contacts between the NAC and the C-terminus. At both pH values, transient contacts between the N- and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral- and low-pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary-structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral- and low-pH ensembles suggests that the change in long-range interactions between the low- and neutral-pH ensembles, the compaction of the C-terminal region at low pH, and the uneven distribution of charges across the sequence are key to faster aggregation.     

The jigsaw puzzle model: search for conformational specificity in protein interiors

The jigsaw puzzle model postulates that the predominant factor relating primary sequence to three-dimensional fold lies in the stereospecific packing of interdigitating side-chains within densely packed protein interiors. An attempt has been made to check the validity of the model by means of a surface complementarity function. Out of a database of 100 highly resolved protein structures the contacts between buried hydrophobic residues (Leu, Ile, Val, Phe) and their neighbours have been categorized in terms of the extent of side-chain surface area involved in a contact (overlap) and their steric fit (Sm). The results show that the majority of contacts between a buried residue and its immediate neighbours (side-chains) are of high steric fit and in the case of extended overlap at least one of the angular parameters characterizing interresidue geometry to have pronounced deviation from a random distribution, estimated by chi(2). The calculations thus tend to support the "jigsaw puzzle" model in that 75-85% of the contacts involving hydrophobic residues are of high surface complementarity, which, coupled to high overlap, exercise fairly stringent constraints over the possible geometrical orientations between interacting residues. These constraints manifest in simple patterns in the distributions of orientational angles. Approximately 60-80% of the buried side-chain surface packs against neighbouring side-chains, the rest interacting with main-chain atoms. The latter partition of the surface maintains an equally high steric fit (relative to side-chain contacts) emphasizing a non-trivial though secondary role played by main-chain atoms in interior packing. The majority of this class of contacts, though of high complementarity, is of reduced overlap. All residues whether hydrophobic or polar/charged show similar surface complementarity measures upon burial, indicating comparable competence of all amino acids in packing effectively with their atomic environments. The specificity thus appears to be distributed over the entire network of contacts within proteins. The study concludes with a proposal to classify contacts as specific and non-specific (based on overlap and fit), with the former perhaps contributing more to the specificity between sequence and fold than the latter.     

The density and refractive index of adsorbing protein layers

The structure of the adsorbing layers of native and denatured proteins (fibrinogen, gamma-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO(2) and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO(2) surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces.     

The effects of antimicrotubule agents on cell motility in fibroblast aggregates

Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in fibroblast motility in the three-dimensional aggregates.     

Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.

We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.     

Hydrophobic potential of mean force as a solvation function for protein structure prediction

We have developed a solvation function that combines a Generalized Born model for polarization of protein charge by the high dielectric solvent, with a hydrophobic potential of mean force (HPMF) as a model for hydrophobic interaction, to aid in the discrimination of native structures from other misfolded states in protein structure prediction. We find that our energy function outperforms other reported scoring functions in terms of correct native ranking for 91% of proteins and low Z scores for a variety of decoy sets, including the challenging Rosetta decoys. This work shows that the stabilizing effect of hydrophobic exposure to aqueous solvent that defines the HPMF hydration physics is an apparent improvement over solvent-accessible surface area models that penalize hydrophobic exposure. Decoys generated by thermal sampling around the native-state basin reveal a potentially important role for side-chain entropy in the future development of even more accurate free energy surfaces.