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.     

Structural analysis of residues involving cation-pi interactions in different folding types of membrane proteins

Cation-pi interactions play an important role to the stability of protein structures. In our earlier work, we have analyzed the influence and energetic contribution of cation-pi interactions in three-dimensional structures of membrane proteins. In this work, we investigate the characteristic features of residues that are involved in cation-pi interactions. We have computed several parameters, such as surrounding hydrophobicity, number of long-range contacts, conservation score and normalized B-factor for all these residues and identified their location, whether in the membrane or at surface. We found that the cation-pi interactions are mainly formed by long-range interactions. The cationic residues involved in cation-pi interactions have higher surrounding hydrophobicity than their average values in the whole dataset and an opposite trend is observed for aromatic residues. In transmembrane helical proteins, except Phe, all other residues that are responsible for cation-pi interactions are highly conserved with other related protein sequences whereas in transmembrane strand proteins, an appreciable conservation is observed only for Arg. The analysis on the flexibility of residues reveals that the cation-pi interaction forming residues are more stable than other residues. The results obtained in the present study would be helpful to understand the role of cation-pi interactions in the structure and folding of membrane proteins.     

Investigation of an Anomalously Accelerating Substitution in the Folding of a Prototypical Two-State Protein

The folding rates of two-state single-domain proteins are generally resistant to small-scale changes in amino acid sequence. For example, having surveyed here over 700 single-residue substitutions in 24 well-characterized two-state proteins, we find that the majority (55%) of these substitutions affect folding rates by less than a factor of 2, and that only 9% affect folding rates by more than a factor of 8. Among those substitutions that significantly affect folding rates, we find that accelerating substitutions are an order of magnitude less common than those that decelerate the process. One of the most extreme outliers in this data set, an arginine-to-phenylalanine substitution at position 48 (R48F) of chymotrypsin inhibitor 2 (CI2), accelerates the protein's folding rate by a factor of 36 relative to that of the wild-type protein and is the most accelerating substitution reported to date in a two-state protein. In order to better understand the origins of this anomalous behavior, we have characterized the kinetics of multiple additional substitutions at this position. We find that substitutions at position 48 in CI2 fall into two distinct classes. The first, comprising residues that ablate the charge of the wild-type arginine but retain the hydrophobicity of its alkane chain, accelerate folding by at least 10-fold. The second class, comprising all other residues, produces folding rates within a factor of two of the wild-type rate. A significant positive correlation between hydrophobicity and folding rate across all of the residues we have characterized at this position suggests that the hydrophobic methylene units of the wild-type arginine play a significant role in stabilizing the folding transition state. Likewise, studies of the pH dependence of the histidine substitution indicate a strong correlation between folding rate and charge state. Thus, mutations that ablate the arginine's positive charge while retaining the hydrophobic contacts of its methylene units tend to dramatically accelerate folding. Previous studies have suggested that arginine 48 plays an important functional role in CI2, which may explain why it is highly conserved despite the anomalously large deceleration it produces in the folding of this protein.     

Correlation of sequence hydrophobicities measures similarity in three-dimensional protein structure

The degree of similarity in the three-dimensional structures of two proteins can be examined by comparing the patterns of hydrophobicity found in their amino acid sequences. Each type of amino acid residue is assigned a numerical hydrophobicity, and the correlation coefficient rH is computed between all pairs of residues in the two sequences. In tests on sequences from two properly aligned proteins of similar three-dimensional structures, rH is found in the range 0.3 to 0.7. Improperly aligned sequences or unrelated sequences give rH near zero. By considering the observed frequency of amino acid replacements among related structures, a set of optimal matching hydrophobicities (OMHs) was derived. With this set of OMHs, significant correlation coefficients are calculated for similar three-dimensional structures, even though the two sequences contain few identical residues. An example is the two similar folding domains of rhodanese (rH = 0.5). Predictions are made of similar three-dimensional structures for the alpha and beta chains of the various phycobiliproteins, and for delta hemolysin and melittin.     

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.     

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.     

Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: application of long-range order to folding rate prediction.

The contact order is believed to be an important factor for understanding protein folding mechanisms. In our earlier work, we have shown that the long-range interactions play a vital role in protein folding. In this work, we analyzed the contribution of long-range contacts to determine the folding rate of two-state proteins. We found that the residues that are close in space and are separated by at least ten to 15 residues in sequence are important determinants of folding rates, suggesting the presence of a folding nucleus at an interval of approximately 25 residues. A novel parameter "long-range order" has been proposed to predict protein folding rates. This parameter shows as good a relationship with the folding rate of two-state proteins as contact order. Further, we examined the minimum limit of residue separation to determine the long-range contacts for different structural classes. We observed an excellent correlation between long-range order and folding rate for all classes of globular proteins. We suggest that in mixed-class proteins, a larger number of residues can serve as folding nuclei compared to all-alpha and all-beta proteins. A simple statistical method has been developed to predict the folding rates of two-state proteins using the long-range order that produces an agreement with experimental results that is better or comparable to other methods in the literature.     

Locating the stabilizing residues in (alpha/beta)8 barrel proteins based on hydrophobicity, long-range interactions, and sequence conservation

In nature, 1 out of every 10 proteins has an (alpha/beta)(8) (TIM)-barrel fold, and in most cases, pairwise comparisons show no sequence similarity between them. Hence, delineating the key residues that induce very different sequences to share a common fold is important for understanding the folding and stability of TIM-barrel domains. In this work, we propose a new consensus approach for locating these stabilizing residues based on long-range interactions, hydrophobicity, and conservation of amino acid residues. We have identified 957 stabilizing residues in 63 proteins from a nonredundant set of 71 TIM-barrel domains. Most of these residues are located in the 8-stranded beta-sheet, with nearly one half of them oriented toward the interior of the barrel and the other half oriented toward the surrounding alpha-helices. Several stabilizing residues are found in the N- and C-terminal loops, whereas very few appear in the alpha-helices that surround the internal beta-sheet. Further, these 957 residues are placed in 434 stabilizing segments of various sizes, and each domain contains 1-10 of these segments. We found that 8 segments per domain is the most abundant one, and two thirds of the proteins have 7-9 stabilizing segments. Finally, we verified the identified residues with experimental temperature factors and found that these residues are among the ones with less mobility in the considered proteins. We suggest that our new protocol serves as a powerful tool to identify the stabilizing residues in TIM-barrel domains, which can be used as potential candidates for studying protein folding and stability by means of protein engineering experiments.     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

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.     

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.     

Exploring the kinetic requirements for enhancement of protein folding rates in the GroEL cavity

The chaperonin system, GroEL and GroES of Escherichia coli enable certain proteins to fold under conditions when spontaneous folding is prohibitively slow as to compete with other non-productive channels such as aggregation. We investigated the plausible mechanisms of GroEL-mediated folding using simple lattice models. In particular, we have investigated protein folding in a confined environment, such as those offered by the GroEL, to decipher whether rate and yield enhancement can occur when the substrate protein is allowed to fold within the cavity of the chaperonins. The GroEL cavity is modeled as a cubic box and a simple bead model is used to represent the substrate chain. We consider three distinct characteristic of the confining environment. First, the cavity is taken to be a passive Anfinsen cage in which the walls merely reduce the available conformation space. We find that at temperatures when the native conformation is stable, the folding rate is retarded in the Anfinsen cage. We then assumed that the interior of the wall is hydrophobic. In this case the folding times exhibit a complex behavior. When the strength of the interaction between the polypeptide chain and the cavity is too strong or too weak we find that the rates of folding are retarded compared to spontaneous folding. There is an optimum range of the interaction strength that enhances the rates. Thus, above this value there is an inverse correlation between the folding rates and the strength of the substrate-cavity interactions. The optimal hydrophobic walls essentially pull the kinetically trapped states which leads to a smoother the energy landscape. It is known that upon addition of ATP and GroES the interior cavity of GroEL offers a hydrophilic-like environment to the substrate protein. In order to mimic this within the context of the dynamic Anfinsen cage model, we allow for changes in the hydrophobicity of the walls of the cavity. The duration for which the walls remain hydrophobic during one cycle of ATP hydrolysis is allowed to vary. These calculations show that frequent cycling of the wall hydrophobicity can dramatically reduce the folding times and increase the yield as well under non-permissive conditions. Examination of the structures of the substrate proteins before and after the change in hydrophobicity indicates that there is global unfolding involved. In addition, it is found that a fraction of the molecules kinetically partition to the native state in accordabce with the iterative annealing mechanism. Thus, frequent "unfoldase" activity of chaperonins leading to global unfolding of the polypeptide chain results in enhancement of the folding rates and yield of the folded protein. We suggest that chaperonin efficiency can be greatly enhanced if the cycling time is reduced. The calculations are used to interpret a few experiments on chaperonin-mediated protein folding.     

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.     

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.     

Diffusion-collision of foldons elucidates the kinetic effects of point mutations and suggests control strategies of the folding process of helical proteins

In this article we use mutation studies as a benchmark for a minimal model of the folding process of helical proteins. The model ascribes a pivotal role to the collisional dynamics of a few crucial residues (foldons) and predicts the folding rates by exploiting information drawn from the protein sequence. We show that our model rationalizes the effects of point mutations on the kinetics of folding. The folding times of two proteins and their mutants are predicted. Stability and location of foldons have a critical role as the determinants of protein folding. This allows us to elucidate two main mechanisms for the kinetic effects of mutations. First, it turns out that the mutations eliciting the most notable effects alter protein stability through stabilization or destabilization of the foldons. Secondly, the folding rate is affected via a modification of the foldon topology by those mutations that lead to the birth or death of foldons. The few mispredicted folding rates of some mutants hint at the limits of the current version of the folding model proposed in the present article. The performance of our folding model declines in case the mutated residues are subject to strong long-range forces. That foldons are the critical targets of mutation studies has notable implications for design strategies and is of particular interest to address the issue of the kinetic regulation of single proteins in the general context of the overall dynamics of the interactome.     

Outlining folding nuclei in globular proteins

Our theoretical approach for prediction of folding/unfolding nuclei in three-dimensional protein structures is based on a search for free energy saddle points on networks of protein unfolding pathways. Under some approximations, this search is performed rapidly by dynamic programming and results in prediction of Phi values, which can be compared with those found experimentally. In this study, we optimize some details of the model (specifically, hydrogen atoms are taken into account in addition to heavy atoms), and compare the theoretically obtained and experimental Phi values (which characterize involvement of residues in folding nuclei) for all 17 proteins, where Phi values are now known for many residues. We show that the model provides good Phi value predictions for proteins whose structures have been determined by X-ray analysis (the average correlation coefficient is 0.65), with a more limited success for proteins whose structures have been determined by NMR techniques only (the average correlation coefficient is 0.34), and that the transition state free energies computed from the same model are in a good anticorrelation with logarithms of experimentally measured folding rates at mid-transition (the correlation coefficient is -0.73).     

Are membrane proteins "inside-out" proteins?

One of the central paradigms of structural biology is that membrane proteins are "inside-out" proteins, in that they have a core of polar residues surrounded by apolar residues. This is the reverse of the characteristics found in water-soluble proteins. We have decided to test this paradigm, now that sufficient numbers of transmembrane alpha-helical structures are accessible to statistical analysis. We have analyzed the correlation between accessibility and hydrophobicity of both individual residues and complete helices. Our analyses reveal that hydrophobicity of residues in a transmembrane helical bundle does not correlate with any preferred location and that the hydrophilic vector of a helix is a poor indicator of the solvent exposed face of a helix. Neither polar nor hydrophobic residues show any bias for the exterior or the interior of a transmembrane domain. As a control, analysis of water-soluble helical bundles performed in a similar manner has yielded clear correlations between hydrophobicity and accessibility. We therefore conclude that, based on the data set used, membrane proteins as "inside-out" proteins is an unfounded notion, suggesting that packing of alpha-helices in membranes is better understood by maximization of van der Waal's forces, rather than by a general segregation of hydrophobicities driven by lipid exclusion.     

Amino acid architecture and the distribution of polar atoms on the surfaces of proteins

We propose that a necessary condition for a protein to be soluble is the absence of large hydrophobic patches on its solvent-accessible surface, which can cause aggregation to occur. We note that the polar nature of the backbone of all amino acids guarantees a minimum polar content and hence can interrupt such patches. As a result, a carefully conserved detailed atomic placement of residues on the protein surface is not necessary for solubility. In order to demonstrate this, we construct a measure based on the average hydrophobicity of a simply defined patch. We use this measurement to compare surfaces that exhibit a clear difference in their solubility properties, namely, a) the solvent accessible surfaces for a set of homo-dimers and the surfaces buried in their interfaces and b) for a set of monomers the surfaces of fragments of secondary structure which are solvent accessible/inaccessible. Having demonstrated a difference in the first set of distributions, we characterize the solvent accessible surfaces of monomeric proteins. To test if cooperative behavior occurs between the atoms for these surfaces, we construct a set of randomized surfaces, which obey a very simple stereochemical constraint. We find that the observed and randomized distributions are much more similar than the previous sets we examined. This implies that while surfaces of soluble proteins must have sufficient polar content, the relative placement of atoms of one amino acid with respect to the atoms of neighboring amino acid need not be finely tuned, which provides an innate robustness for protein design and folding.     

Importance of hydrophobic cluster formation through long-range contacts in the folding transition state of two-state proteins

Understanding the folding pathways of proteins is a challenging task. The Phi value approach provides a detailed understanding of transition-state structures of folded proteins. In this work, we have computed the hydrophobicity associated with each residue in the folded state of 16 two-state proteins and compared the Phi values of each mutant residue. We found that most of the residues with high Phi value coincide with local maximum in surrounding hydrophobicity, or have nearby residues that show such maximum in hydrophobicity, indicating the importance of hydrophobic interactions in the transition state. We have tested our approach to different structural classes of proteins, such as alpha-helical, SH3 domains of all-beta proteins, beta-sandwich, and alpha/beta proteins, and we observed a good agreement with experimental results. Further, we have proposed a hydrophobic contact network pattern to relate the Phi values with long-range contacts, which will be helpful to understand the transition-state structures of folded proteins. The present approach could be used to identify potential hydrophobic clusters that may form through long-range contacts during the transition state.