Nanometric design of extraordinary hydrophobic-induced pKa shifts for aspartic acid: Relevance to protein mechanisms

Commonly a key element enabling proteins to function is an amino acid residue or residues with functional side chains having shifted pKa values. This article reports the results on a set of protein-based polymers (model proteins) that exhibit hydrophobic folding and assembly transitions, and that have been designed for the purpose of achieving large hydrophobic-induced pKa shifts by selectively replacing Val residues by Phe residues. The high molecular weight polypentapeptides, actually poly (tricosapeptides) with six varied pentamers in fixed sequence, were designed and synthesized to have the same amino acid compositions but different proximities between a single aspartic acid residue and 5 Phe residues per 30 residues. With the 5 Phe residues distal from the Asp residue, the observed pKa shift was 2.9 when compared to the Val-containing reference. With the 5 Phe residues within 1 nm of the Asp residue, the pKa shift was 6.2. This represents a free energy of interaction of 8 kcal/moles. To our knowledge, this is the largest pKa shift documented for an Asp residue in a polypeptide– or protein–water system. Data are reviewed that do not support the usual electrostatic arguments for pKa shifts of charge–charge repulsion and/or unfavorable ion self-energies arising from displacement of water by hydrophobic moieties, but rather the data are interpreted to indicate the presence of an apolar–polar repulsive free energy of hydration, which results from a potentially highly cooperative competition between apolar and polar species for hydration. © 1994 John Wiley & Sons, Inc.     

Hydrophobicity scale for proteins based on inverse temperature transitions.

In general, proteins fold with hydrophobic residues buried, away from water. Reversible protein folding due to hydrophobic interactions results from inverse temperature transitions where folding occurs on raising the temperature. Because homoiothermic animals constitute an infinite heat reservoir, it is the transition temperature, Tt, not the endothermic heat of the transition, that determines the hydrophobically folded state of polypeptides at body temperature. Reported here is a new hydrophobicity scale based on the values of Tt for each amino acid residue as a guest in a natural repeating peptide sequence, the high polymers of which exhibit reversible inverse temperature transitions. Significantly, a number of ways have been demonstrated for changing Tt such that reversibly lowering Tt from above to below physiological temperature becomes a means of isothermally and reversibly driving hydrophobic folding. Accordingly, controlling Tt becomes a mechanism whereby proteins can be induced to carry out isothermal free energy transduction.     

Reduction-driven polypeptide folding by the delta Tt mechanism.

Poly(Gly-Val-Gly-Val-Pro), i.e., poly(GVGVP), exhibits composition and solute dependence of Tt, the temperature of the inverse temperature transition at which hydrophobic folding and assembly occur on raising the temperature. Importantly, a means whereby the value of Tt is lowered from above to below the working temperature becomes an isothermal means of driving folding and assembly, i.e., of achieving free energy transduction. Using poly[0.73(GVGVP),0.27(GK[NMeN]GVP)] where [NMeN] indicates N-methyl nicotinamide attached to the epsilon-NH2 of the Lys(K) residue, chemical and electrochemical reductions are found to remarkably lower the value of Tt; reduction can drive hydrophobic folding and assembly as effectively as decreasing ionization. Changing the redox state of a protein becomes yet another means of achieving free energy transduction by the delta Tt mechanism.     

Protein‐spanning water networks and implications for prediction of protein–protein interactions mediated through hydrophobic effects

Hydrophobic effects, often conflated with hydrophobic forces, are implicated as major determinants in biological association and self‐assembly processes. Protein–protein interactions involved in signaling pathways in living systems are a prime example where hydrophobic effects have profound implications. In the context of protein–protein interactions, a priori knowledge of relevant binding interfaces (i.e., clusters of residues involved directly with binding interactions) is difficult. In the case of hydrophobically mediated interactions, use of hydropathy‐based methods relying on single residue hydrophobicity properties are routinely and widely used to predict propensities for such residues to be present in hydrophobic interfaces. However, recent studies suggest that consideration of hydrophobicity for single residues on a protein surface require accounting of the local environment dictated by neighboring residues and local water. In this study, we use a method derived from percolation theory to evaluate spanning water networks in the first hydration shells of a series of small proteins. We use residue‐based water density and single‐linkage clustering methods to predict hydrophobic regions of proteins; these regions are putatively involved in binding interactions. We find that this simple method is able to predict with sufficient accuracy and coverage the binding interface residues of a series of proteins. The approach is competitive with automated servers. The results of this study highlight the importance of accounting of local environment in determining the hydrophobic nature of individual residues on protein surfaces. Proteins 2014; 82:3312–3326. © 2014 Wiley Periodicals, Inc.     

Analysis on folding of misgurin using two-dimensional HP model

Misgurin is an antimicrobial peptide from the loach, while the hydrophobic-polar (HP) model is a way to study the folding conformations and native states in peptide and protein although several amino acids cannot be classified either hydrophobic or polar. Practically, the HP model requires extremely intensive computations, thus it has yet to be used widely. In this study, we use the two-dimensional HP model to analyze all possible folding conformations and native states of misgurin with conversion of natural amino acids according to the normalized amino acid hydrophobicity index as well as the shortest benchmark HP sequence. The results show that the conversion of misgurin into HP sequence with glycine as hydrophobic amino acid at pH 2 has 1212 folding conformations with the same native state of minimal energy -6; the conversion of glycine as polar amino acid at pH 2 has 13,386 folding conformations with three native states of minimal energy -5; the conversion of glycine as hydrophobic amino acid at pH 7 has 2538 folding conformations with three native states of minimal energy -5; and the conversion of glycine as polar amino acid at pH 7 has 12,852 folding conformations with three native states of minimal energy -4. Those native states can be ranked according to the normalized amino acid hydrophobicity index. The detailed discussions suggest two ways to modify misgurin.     

Protein Structure Classification Based on Conserved Hydrophobic Residues

Protein folding is frequently guided by local residue interactions that form clusters in the protein core. The interactions between residue clusters serve as potential nucleation sites in the folding process. Evidence postulates that the residue interactions are governed by the hydrophobic propensities that the residues possess. An array of hydrophobicity scales has been developed to determine the hydrophobic propensities of residues under different environmental conditions. In this work, we propose a graph-theory-based data mining framework to extract and isolate protein structural features that sustain invariance in evolutionary-related proteins, through the integrated analysis of five well-known hydrophobicity scales over the 3D structure of proteins. We hypothesize that proteins of the same homology contain conserved hydrophobic residues and exhibit analogous residue interaction patterns in the folded state. The results obtained demonstrate that discriminatory residue interaction patterns shared among proteins of the same family can be employed for both the structural and the functional annotation of proteins. We obtained on the average 90 percent accuracy in protein classification with a significantly small feature vector compared to previous results in the area. This work presents an elaborate study, as well as validation evidence, to illustrate the efficacy of the method and the correctness of results reported.     

Statistical distribution of hydrophobic residues along the length of protein chains. Implications for protein folding and evolution.

We consider in this paper the statistical distribution of hydrophobic residues along the length of protein chains. For this purpose we used a binary hydrophobicity scale which assigns hydrophobic residues a value of one and non-hydrophobes a value of zero. The resulting binary sequences are tested for randomness using the standard run test. For the majority of the 5,247 proteins examined, the distribution of hydrophobic residues along a sequence cannot be distinguished from that expected for a random distribution. This suggests that (a) functional proteins may have originated from random sequences, (b) the folding of proteins into compact structures may be much more permissive with less sequence specificity than previously thought, and (c) the clusters of hydrophobic residues along chains which are revealed by hydrophobicity plots are a natural consequence of a random distribution and can be conveniently described by binomial statistics.     

Gauss-function-Based model of hydrophobicity density in proteins

The model adopting the three-dimensional Gauss function to express the hydrophobicity distribution in proteins is presented in this paper. The tendency to create the hydrophobic center during protein folding is expressed in form of an external force field of the form of three-dimensional Gauss function which directs the folding polypeptide to locate the hydrophobic residues in a central part of the molecule and hydrophilic ones exposed toward the molecular surface. The decrease of the differences between hydrophobicity distribution as it appears at each step of the folding simulation and the expected hydrophobicity distribution (three-dimensional Gauss function) is the convergence criterion together with traditional non-bonding interaction optimization. The model was applied to fold the hypothetical membrane protein (target protein in CASP6) TA0354_69_121 from Thermoplasma acidophilum.     

Frequencies of hydrophobic and hydrophilic runs and alternations in proteins of known structure

Patterns of alternation of hydrophobic and polar residues are a profound aspect of amino acid sequences, but a feature not easily interpreted for soluble proteins. Here we report statistics of hydrophobicity patterns in proteins of known structure in a current protein database as compared with results from earlier, more limited structure sets. Previous studies indicated that long hydrophobic runs, common in membrane proteins, are underrepresented in soluble proteins. Long runs of hydrophobic residues remain significantly underrepresented in soluble proteins, with none longer than 16 residues observed. These long runs most commonly occur as buried alpha helices, with extended hydrophobic strands less common. Avoiding aggregation of partially folded intermediates during intracellular folding remains a viable explanation for the rarity of long hydrophobic runs in soluble proteins. Comparison between database editions reveals robustness of statistics on aqueous proteins despite an approximately twofold increase in nonredundant sequences. The expanded database does now allow us to explain several deviations of hydrophobicity statistics from models of random sequence in terms of requirements of specific secondary structure elements. Comparison to prior membrane-bound protein sequences, however, shows significant qualitative changes, with the average hydrophobicity and frequency of long runs of hydrophobic residues noticeably increasing between the database editions. These results suggest that the aqueous proteins of solved structure may represent an essentially complete sample of the universe of aqueous sequences, while the membrane proteins of known structure are not yet representative of the universe of membrane-associated proteins, even by relatively simple measures of hydrophobic patterns.     

Hydrophobic environment is a key factor for the stability of thermophilic proteins

The stability of thermophilic proteins has been viewed from different perspectives and there is yet no unified principle to understand this stability. It would be valuable to reveal the most important interactions for designing thermostable proteins for such applications as industrial protein engineering. In this work, we have systematically analyzed the importance of various interactions by computing different parameters such as surrounding hydrophobicity, inter‐residue interactions, ion‐pairs and hydrogen bonds. The importance of each interaction has been determined by its predicted relative contribution in thermophiles versus the same contribution in mesophilic homologues based on a dataset of 373 protein families. We predict that hydrophobic environment is the major factor for the stability of thermophilic proteins and found that 80% of thermophilic proteins analyzed showed higher hydrophobicity than their mesophilic counterparts. Ion pairs, hydrogen bonds, and interaction energy are also important and favored in 68%, 50%, and 62% of thermophilic proteins, respectively. Interestingly, thermophilic proteins with decreased hydrophobic environments display a greater number of hydrogen bonds and/or ion pairs. The systematic elimination of mesophilic proteins based on surrounding hydrophobicity, interaction energy, and ion pairs/hydrogen bonds, led to correctly identifying 95% of the thermophilic proteins in our analyses. Our analysis was also applied to another, more refined set of 102 thermophilic–mesophilic pairs, which again identified hydrophobicity as a dominant property in 71% of the thermophilic proteins. Further, the notion of surrounding hydrophobicity, which characterizes the hydrophobic behavior of residues in a protein environment, has been applied to the three‐dimensional structures of elongation factor‐Tu proteins and we found that the thermophilic proteins are enriched with a hydrophobic environment. The results obtained in this work highlight the importance of hydrophobicity as the dominating characteristic in the stability of thermophilic proteins, and we anticipate this will be useful in our attempts to engineering thermostable proteins. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins

Hydrophobicity is thought to be one of the primary forces driving the folding of proteins. On average, hydrophobic residues occur preferentially in the core, whereas polar residues tend to occur at the surface of a folded protein. By analyzing the known protein structures, we quantify the degree to which the hydrophobicity sequence of a protein correlates with its pattern of surface exposure. We have assessed the statistical significance of this correlation for several hydrophobicity scales in the literature, and find that the computed correlations are significant but far from optimal. We show that this less than optimal correlation arises primarily from the large degree of mutations that naturally occurring proteins can tolerate. Lesser effects are due in part to forces other than hydrophobicity, and we quantify this by analyzing the surface-exposure distributions of all amino acids. Lastly, we show that our database findings are consistent with those found from an off-lattice hydrophobic-polar model of protein folding.     

Empirical lipid propensities of amino acid residues in multispan alpha helical membrane proteins

Characterizing the interactions between amino acid residues and lipid molecules is important for understanding the assembly of transmembrane helices and for studying membrane protein folding. In this study we develop TMLIP (TransMembrane helix-LIPid), an empirically derived propensity of individual residue types to face lipid membrane based on statistical analysis of high-resolution structures of membrane proteins. Lipid accessibilities of amino acid residues within the transmembrane (TM) region of 29 structures of helical membrane proteins are studied with a spherical probe of radius of 1.9 A. Our results show that there are characteristic preferences for residues to face the headgroup region and the hydrocarbon core region of lipid membrane. Amino acid residues Lys, Arg, Trp, Phe, and Leu are often found exposed at the headgroup regions of the membrane, where they have high propensity to face phospholipid headgroups and glycerol backbones. In the hydrocarbon core region, the strongest preference for interacting with lipids is observed for Ile, Leu, Phe and Val. Small and polar amino acid residues are usually buried inside helical bundles and are strongly lipophobic. There is a strong correlation between various hydrophobicity scales and the propensity of a given residue to face the lipids in the hydrocarbon region of the bilayer. Our data suggest a possibly significant contribution of the lipophobic effect to the folding of membrane proteins. This study shows that membrane proteins have exceedingly apolar exteriors rather than highly polar interiors. Prediction of lipid-facing surfaces of boundary helices using TMLIP1 results in a 54% accuracy, which is significantly better than random (25% accuracy). We also compare performance of TMLIP with another lipid propensity scale, kPROT, and with several hydrophobicity scales using hydrophobic moment analysis.     

Structural transitions of confined model proteins: molecular dynamics simulation and experimental validation

Proteins fold in a confined space not only in vivo, i.e., folding assisted by molecular chaperons and chaperonins in a crowded cellular medium, but also in vitro as in production of recombinant proteins. Despite extensive work on protein folding in bulk, little is known about how and to what extent the thermodynamics and kinetics of protein folding are altered by confinement. In this work, we use a Gō-like off-lattice model to investigate the folding and stability of an all beta-sheet protein in spherical cages of different sizes and surface hydrophobicity. We find whereas extreme confinement inhibits correct folding, a hydrophilic cage stabilizes the protein due to restriction of the unfolded configurations. In a hydrophobic cage, however, strong attraction from the cage surface destabilizes the confined protein because of competition between self-aggregation and adsorption of hydrophobic residues. We show that the kinetics of protein collapse and folding is strongly correlated with both the cage size and the surface hydrophobicity. It is demonstrated that a cage of moderate size and hydrophobicity optimizes both the folding yield and kinetics of structural transitions. To support the simulation results, we have also investigated the refolding of hen-egg lysozyme in the presence of cetyltrimethylammoniumbromide (CTAB) surfactants that provide an effective confinement of the proteins by micellization. The influence of the surfactant hydrophobicity on the structural and biological activity of the protein is determined with circular dichroism spectrum, fluorescence emission spectrum, and biological activity assay. It is shown that, as predicted by coarse-grained simulations, CTAB micelles facilitate the collapse of denatured lysozyme, whereas the addition of beta-cyclodextrin-grafted-PNIPAAm, a weakly hydrophobic stripper, dissociates CTAB micelles and promotes the conformational rearrangement and thereby gives an improved recovery of lysozyme activity.     

Folding-unfolding energy change of a simple sphere model protein and an energy landscape of the folding process

We have calculated the free energy of a spherical model of a protein or part of a protein generated in the way of protein folding. Two spherical models are examined; one is a homogeneous model consisting of only one residue type—hydrophobic. The other is a heterogeneous model consisting of two residue types—strong hydrophobic and weak hydrophobic. Both models show a folding transition state, and the latter model reproduces the trend of the experimental folded-unfolded energy change. The heterogeneous model suggests that in the folding process of a protein of more than 70 residues, a specific region of the protein folds first to form a stable region, then the other residues follow the folding process. The energy landscape of folding of a small protein is approximately a funnel model, whereas a flatter energy landscape is suggested for larger proteins of more than 55–70 residues. Proteins 33:408–416, 1998. © 1998 Wiley-Liss, Inc.     

Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations

Understanding the solvation of amino acids in biomembranes is an important step to better explain membrane protein folding. Several experimental studies have shown that polar residues are both common and important in transmembrane segments, which means they have to be solvated in the hydrophobic membrane, at least until helices have aggregated to form integral proteins. In this work, we have used computer simulations to unravel these interactions on the atomic level, and classify intramembrane solvation properties of amino acids. Simulations have been performed for systematic mutations in poly-Leu helices, including not only each amino acid type, but also every z-position in a model helix. Interestingly, many polar or charged residues do not desolvate completely, but rather retain hydration by snorkeling or pulling in water/headgroups--even to the extent where many of them exist in a microscopic polar environment, with hydration levels corresponding well to experimental hydrophobicity scales. This suggests that even for polar/charged residues a large part of solvation cost is due to entropy, not enthalpy loss. Both hydration level and hydrogen bonding exhibit clear position-dependence. Basic side chains cause much less membrane distortion than acidic, since they are able to form hydrogen bonds with carbonyl groups instead of water or headgroups. This preference is supported by sequence statistics, where basic residues have increased relative occurrence at carbonyl z-coordinates. Snorkeling effects and N-/C-terminal orientation bias are directly observed, which significantly reduces the effective thickness of the hydrophobic core. Aromatic side chains intercalate efficiently with lipid chains (improving Trp/Tyr anchoring to the interface) and Ser/Thr residues are stabilized by hydroxyl groups sharing hydrogen bonds to backbone oxygens.     

Apparent pKa shifts of titratable residues at high denaturant concentration and the impact on protein stability

Urea and guanidine-hydrochloride (GdnHCl) are frequently used for protein denaturation in order to determine the Gibbs free energy of folding and kinetic folding/unfolding parameters. Constant pH value is applied in the folding/unfolding experiments at different denaturant concentrations and steady protonation state of titratable groups is assumed in the folded and unfolded protein, respectively. The apparent side-chain pKa values of Asp, Glu, His and Lys in the absence and presence of 6 M urea and GdnHCl, respectively, have been determined by 1H-NMR. pKa values of all four residues are up-shifted by 0.3-0.5 pH units in presence of 6 M urea by comparison with pKa values of the residues dissolved in water. In the presence of 6 M GdnHCl, pKa values are down-shifted by 0.2-0.3 pH units in the case of acidic and up-shifted by 0.3-0.5 pH units in the case of basic residues. Shifted pKa values in the presence of denaturant may have a pronounced effect on the outcome of the protein stability obtained from denaturant unfolding experiments.     

Importance of mutant position in Ramachandran plot for predicting protein stability of surface mutations

Understanding the mechanisms by which mutations affect protein stability is one of the most important problems in molecular biology. In this work, we analyzed the relationship between changes in protein stability caused by surface mutations and changes in 49 physicochemical, energetic, and conformational properties of amino acid residues. We found that the hydration entropy was the major contributor to the stability of surface mutations in helical segments; other properties responsible for size and volume of molecule also correlated significantly with stability. Classification of coil mutations based on their locations in the (phi-psi) map improved the correlation significantly, demonstrating the existence of a relationship between stability and strain energy, which indicates that the role of strain energy is very important for the stability of surface mutations. We observed that the inclusion of sequence and structural information raised the correlation, indicating the influence of surrounding residues on the stability of surface mutations. Further, we examined the previously reported "inverse relationship" between stability and hydrophobicity, and observed that the inverse hydrophobic effect was generally applicable only to coil mutations. The present study leads to a simple method for predicting protein stability changes caused by amino acid substitutions, which will be useful for protein engineering in designing novel proteins with increased stability and altered function.