Buried Residues Research Articles

M2 amphipathic helices facilitate pH-dependent conformational transition in influenza A virus

The matrix-2 (M2) protein from influenza A virus is a tetrameric, integral transmembrane (TM) protein that plays a vital role in viral replication by proton flux into the virus. The His37 tetrad is a pH sensor in the center of the M2 TM helix that activates the channel in response to the low endosomal pH. M2 consists of different regions that are believed to be involved in membrane targeting, packaging, nucleocapsid binding, and proton transport. Although M2 has been the target of many experimental and theoretical studies that have led to significant insights into its structure and function under differing conditions, the main mechanism of proton transport, its conformational dynamics, and the role of the amphipathic helices (AHs) on proton conductance remain elusive. To this end, we have applied explicit solvent constant pH molecular dynamics using the multisite λ-dynamics approach (CpHMDMSλD) to investigate the buried ionizable residues comprehensively and to elucidate their effect on the conformational transition. Our model recapitulates the pH-dependent conformational transition of M2 from closed to open state when the AH domain is included in the M2 construct, revealing the role of the amphipathic helices on this transition and shedding light on the proton-transport mechanism. This work demonstrates the importance of including the amphipathic helices in future experimental and theoretical studies of ion channels. Finally, our work shows that explicit solvent CpHMDMSλD provides a realistic pH-dependent model for membrane proteins.

PPerturb: A Server for Predicting Long-Distance Energetic Couplings and Mutation-Induced Stability Changes in Proteins via Perturbations.

The strength of intraprotein interactions or contact network is one of the dominant factors determining the thermodynamic stabilities of proteins. The nature and the extent of connectivity of this network also play a role in allosteric signal propagation characteristics upon ligand binding to a protein domain. Here, we develop a server for rapid quantification of the strength of an interaction network by employing an experimentally consistent perturbation approach previously validated against a large data set of 375 mutations in 19 different proteins. The web server can be employed to predict the extent of destabilization of proteins arising from mutations in the protein interior in experimentally relevant units. Moreover, coupling distances—a measure of the extent of percolation on perturbation—and overall perturbation magnitudes are predicted in a residue-specific manner, enabling a first look at the distribution of energetic couplings in a protein or its changes upon ligand binding. We show specific examples of how the server can be employed to probe for the distribution of local stabilities in a protein, to examine changes in side chain orientations or packing before and after ligand binding, and to predict changes in stabilities of proteins upon mutations of buried residues. The web server is freely available at http://pbl.biotech.iitm.ac.in/pPerturb and supports recent versions of all major browsers.

Early Metastable Assembly during the Stress-Induced Formation of Worm-like Amyloid Fibrils of Nucleic Acid Binding Domains of TDP-43.

TDP-43 protein travels between the cytosol and the nucleus to perform its nucleic acid binding functions through its two tandem RNA recognition motif domains (TDP-43tRRM). When exposed to various environmental stresses, it forms abnormal aggregates in the cytosol of neurons, which are the hallmarks of amyotrophic lateral sclerosis and other TDP-43 proteinopathies. However, the nature of early structural changes upon stress sensing and the consequent steps during the course of aggregation are not well understood. In this study, we show that under low-pH conditions, mimicking starvation stress, TDP-43tRRM undergoes a conformational opening reaction linked to the protonation of buried ionizable residues and grows into a metastable oligomeric assembly (called the "low-pH form" or the "L form"). In the L form, the protein molecules have disrupted tertiary structure, solvent-exposed hydrophobic patches, and mobile side chains but the native-like secondary structure remains intact. The L form structure is held by weak interactions and has a steep dependence on ionic strength. In the presence of as little as 15 mM KCl, it fully misfolds and further oligomerizes to form a β-sheet rich "β form" in at least two distinct steps. The β form has an ordered, stable structure that resembles worm-like amyloid fibrils. The unstructured regions of the protein gain structure during L ⇌ β conversion. Our results suggest that TDP-43tRRM could function as a stress sensor and support a recent model in which stress sensing during neurodegeneration occurs by assembly of proteins into metastable assemblies that are precursors to the solid aggregates.

Natively oxidized amino acid residues in the spinach PS I-LHC I supercomplex.

Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome b6/f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including O2.-, 1O2, H2O2 and, possibly, •OH;however, the site(s) of ROS production within PS I has been the subject of significant debate. We hypothesize that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in spinach PS I which was isolated from field-grown spinach. The modified residues were identified by high-resolution tandem mass spectrometry. As expected, many of the modified residues lie on the surface of the complex. However, a well-defined group of oxidized residues, both buried and surface-exposed, lead from the chl a' of P700 to the surface of PS I. These residues (PsaB: 609F, 611E, 617M, 619W, 620L, and PsaF: 139L, 142A,143D) may identify a preferred route for ROS, probably 1O2, to egress the complex from the vicinity of P700. Additionally, two buried residues located in close proximity to A1B (PsaB:712H and 714S) were modified, which appearsconsistent with A1B being a source of O2.-. Surprisingly, no oxidatively modified residues were identified in close proximity to the 4Fe-FS clusters FX, FA or FB. These cofactors had been identified as principal targets for ROS damage in the photosystem. Finally, a large number of residues located in the hydrophobic cores of Lhca1-Lhca4 are oxidatively modified. These appear to be the result of 1O2 production by the distal antennae for the photosystem.

HP1 reshapes nucleosome core to promote phase separation of heterochromatin.

Heterochromatin impacts genome function at multiple scales. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. It has been proposed that these diverse functions arise in part from compaction of the underlying chromatin. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3–6. Yet how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we demonstrate that chromatin compaction by the S. pombe HP1 protein, Swi6, results in phase-separated liquid condensates. Remarkably, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs chromatin compaction by Swi6 into liquid droplets. Our results indicate that Swi6 couples oligomerization to the phase separation of chromatin by a counter-intuitive mechanism, namely dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin.

Promotion Effect of Succinimide on Amyloid Fibrillation of Hen Egg-White Lysozyme.

Amyloid fibrillation is closely associated with a series of neurodegenerative diseases. According to that, the intermediate soluble oligomers and protofibrils are more toxic; reducing their concentrations in protein solutions by accelerating fibrillation is believed as a feasible strategy for treatment or remission of the diseases. Using hen egg-white lysozyme (HEWL) as a model protein, the promotion effect of succinimide was revealed by a series of experiments, e.g., atomic force microscopy (AFM), thioflavin T (ThT) fluorescence assay, Far-UV circular dichroism (CD) and Raman spectroscopy, and modeling the effect of succinimide-like derivative intermediates of intramolecular deamidation of the backbone during amyloid fibrillation. The AFM measurement confirmed that succinimide effectively accelerated the morphological changes of HEWL, while at the molecular level, the accelerative transformation of protein secondary structures was also clarified by ThT fluorescence assay and Far-UV CD spectroscopy. The incubation time-dependent Raman spectroscopy further revealed that the direct transformation from α-helices to organized β-sheets occurred upon skipping the intermediate random coils under the action of succinimide. This "bridge" effect of succinimide was attributed to its special influence on disulfide bonds. In the presence of succinimide in protein solutions, the native disulfide bonds of lysozyme could be broken more efficiently and quickly within hydrolysis, resulting in exposure of the buried hydrophobic residues and accelerating the formation of cross β-sheet structures. The present investigation provides very useful information for understanding the effect of intramolecular deamidation on the whole amyloid fibrillation.

Network analysis of dynamically important residues in protein structures mediating ligand-binding conformational changes.

According to the generalized conformational selection model, ligand binding involves the co-existence of at least two conformers with different ligand-affinities in a dynamical equilibrium. Conformational transitions between them should be guaranteed by intramolecular vibrational dynamics associated to each conformation. These motions are, therefore, related to the biological function of a protein. Positions whose mutations are found to alter these vibrations the most can be defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. In a previous study, we have shown that these positions are evolutionarily conserved. They correspond to buried aliphatic residues mostly localized in regular structured regions of the protein like β-sheets and α-helices. In the present paper, we perform a network analysis of these key positions for a large dataset of paired protein structures in the ligand-free and ligand-bound form. We observe that networks of interactions between these key positions present larger and more integrated networks with faster transmission of the information. Besides, networks of residues result that are robust to conformational changes. Our results reveal that the conformational diversity of proteins seems to be guaranteed by a network of strongly interconnected key positions rather than individual residues.

Metal binding and conformational studies of the calcium binding domain of NADPH oxidase 5 reveal its similarity and difference to calmodulin

The superoxide-generating activity of Nox5 is regulated by Ca2+ flux, primarily through its self-contained calcium binding domain (EFD). Upon Ca2+ binding, Nox5’s EFD undergoes a conformational change that exposes its buried hydrophobic residues. Previously, we determined the Ca2+ binding constants of the N-terminal half domain (N-EFD). Here we performed a similar characterization with its C-terminal lobe (C-EFD). Our studies revealed that the binding affinities (Ka’s) of the EFD are in the range of 108–105 M−1 with a strong Ca2+ binding that occurs in the C-EFD. The 3rd Ca2+ binding site also binds Mg2+ (Ka = 4.53 × 103 M−1), where its high Ca2+ binding affinity becomes moderate in cellular conditions. The essential hydrophobic exposure upon metal binding was assessed with the analysis of the 1-anilino-8-naphthalene sulfonate (ANS) interaction via fluorescence and calorimetry. While the ANS fluorescence and binding studies agree with each other in general, the results do not correlate to the actual hydrophobic exposure content. The heat capacity change (ΔCp) of Ca2+ binding for EFD is −24.1 cal/mol.K, while those of N-EFD and C-EFD are −56.3 and −41.6 cal/mol·K, respectively, indicating a significant hydrophobic exposure and polar burial. The latter was confirmed by limited trypsin digestion. The comparison of Nox5’s EFD to calmodulin, including homology modeling, was discussed in the report.Communicated by Ramaswamy H. Sarma

Deciphering the intrinsic properties of fungal proteases in optimizing phytopathogenic interaction

Phytopathogenic fungi secrete a wide range of enzymes to penetrate and colonize host tissues. Of them protease activity is reported to increase disease aggressiveness in the plant. With the aim to explore the reason of the higher infection potential of proteases, we have compared several genomic and proteomic attributes among different hydrolytic enzymes coded by five pathogenic fungal species which are the potent infectious agents of plant. Categorizing the enzymes into four major groups, namely protease, lipase, amylase and cell-wall degraders, we observed that proteases are evolutionary more conserved, have higher expression levels, contain more hydrophobic buried residues, short linear motifs and post-translational modified (PTM) sites than the other three groups of enzymes. Again, comparing these features of protease between pathogenic and non-pathogenic Aspergillus sps, we have hypothesized that protein structural properties could play significant roles in imposing infection potency to the fungal proteases.

Improvement of Selenomonas ruminantium β-xylosidase thermal stability by replacing buried free cysteines via site directed mutagenesis

β-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium β-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results.

Molecular Determinants of Temperature-Sensitive Phenotypes.

Temperature-sensitive (Ts) mutants are important tools for understanding the role of essential gene(s), but their molecular basis is not well understood. We use CcdB ( Controller of Cell Death protein B) as a model system to explore the effects of Ts mutations on protein stability, folding, and ligand binding. Previously isolated Ts CcdB mutants fall broadly into two categories, namely, buried site (<5% accessibility) and active site (involved in DNA gyrase binding). Several mutants from each category were characterized. It was found that buried-site Ts mutants had decreased stability and foldability, higher aggregation propensity, and, in most cases, reduced affinity for gyrase at both permissive and restrictive temperatures. In contrast, exposed, active-site Ts mutants of CcdB exhibited stability either higher than or similar to that of the wild type and weakened inhibition of DNA gyrase function and/or reduced affinity for gyrase at a higher temperature. At all temperatures, Ts mutations at exposed, active-site residues primarily decrease specific activity without affecting protein levels, while Ts mutations at most buried residues decrease both specific activity and protein levels. Ts phenotypes in both cases arise because total activity is decreased below the threshold required for survival at the restrictive temperature but remains above it at the permissive temperatures. For several mutants, Ts phenotypes were ameliorated upon overexpression of the trigger factor chaperone, suggesting that Ts phenotypes may result from mutational effects on in vivo protein folding rather than on protein stability. This study delineates the diverse factors that contribute to Ts phenotypes. These insights can facilitate rational design of Ts mutants.

Relative Binding Affinity Prediction of Charge-Changing Sequence Mutations with FEP in Protein–Protein Interfaces

Building on the substantial progress that has been made in using free energy perturbation (FEP) methods to predict the relative binding affinities of small molecule ligands to proteins, we have previously shown that results of similar quality can be obtained in predicting the effect of mutations on the binding affinity of protein–protein complexes. However, these results were restricted to mutations which did not change the net charge of the side chains due to known difficulties with modeling perturbations involving a change in charge in FEP. Various methods have been proposed to address this problem. Here we apply the co-alchemical water approach to study the efficacy of FEP calculations of charge changing mutations at the protein–protein interface for the antibody–gp120 system investigated previously and three additional complexes. We achieve an overall root mean square error of 1.2 kcal/mol on a set of 106 cases involving a change in net charge selected by a simple suitability filter using side-chain predictions and solvent accessible surface area to be relevant to a biologic optimization project. Reasonable, although less precise, results are also obtained for the 44 more challenging mutations that involve buried residues, which may in some cases require substantial reorganization of the local protein structure, which can extend beyond the scope of a typical FEP simulation. We believe that the proposed prediction protocol will be of sufficient efficiency and accuracy to guide protein engineering projects for which optimization and/or maintenance of a high degree of binding affinity is a key objective.

Experimental Strategy for Engineering pH Switch Proteins

Dysregulation of pH is one of the hallmarks of solid cancerous tumors. An increase in extrusion of acid from tumor cells leads to acidification of the extracellular environment, which in tumors is 0.4 to 0.8 pH units lower than in normal cells. The efficiency and selectivity of proteins (e.g. toxins and antibodies) designed to identify and kill tumor cells would be enhanced if they could be engineered to function as pH switches that are activated/deactivated by small changes in pH in the physiological range. We have demonstrated that buried ionizable residues (Lys, Glu and Asp) are useful for this purpose. Most buried ionizable residues titrate with anomalous pKa values (depressed for Lys and elevated for Glu/Asp) simply by virtue of being buried. This ensures that the residue is neutral when buried in the hydrophobic interior of the protein, consistent with expectations based on the energetic cost of removing a charged moiety from water. The shifts in pKa increase the pH sensitivity of thermodynamic stability. Using staphylococcal nuclease as a test system we engineered many variants with pairs of ionizable groups (Lys or Glu with anomalous pKa values) and demonstrated that this was sufficient to convert this protein into a pH switch that unfolds cooperatively near pH 7. Constructs with pairs of buried Lys residues are folded at high pH and unfolded by decreasing pH and constructs with pairs of buried Glu or Asp are folded at low pH and unfolded by increasing pH. Systematic engineering of several switch proteins with ionizable groups buried in different locations demonstrated that this approach is robust. Based on general thermodynamic principles, this strategy should be transferable to other proteins.

PH-Driven Conformational Reorganization of Proteins: NMR Spectroscopy Study with Buried Lys Residues

The pH sensitivity of proteins is essential for biological energy transduction and for cellular and physiological homeostasis. The thermodynamic basis of pH effects in proteins is well understood but detailed mechanistic understanding of how changes in pH drive conformational reorganization in proteins is not. This was addressed systematically using NMR spectroscopy to characterize pH-dependent conformational changes coupled to the ionization of residues buried in the hydrophobic core of a protein. The study used 25 variants of staphylococcal nuclease with buried Lys residues. The pKa values of 19 of 25 of these internal Lys residues are depressed relative to the normal pKa of Lys in water, some by as much as 5 pH units. This shift in pKa ensures that the ionizable moiety stays neutral at pH values below 10 when it is buried, consistent with what is expected from the unfavorable transfer of a charge from water to a hydrophobic environment. In most cases, ionization of the buried Lys shifts the conformation of the protein into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of reorganization coupled to the ionization of buried Lys residues. The structural details of the conformational response were characterized with backbone chemical shift perturbation analysis, titrations of the buried Nζ atom, and in some cases even X-ray crystallography. At least in this protein, the anomalous pKa values of buried groups are governed by the propensity of the protein to reorganize. These results demonstrate that computational modeling of pH dependent processes in proteins will require prediction of alternative conformational states and accurate calculation of free energies; this remains a significant challenge even under the most favorable circumstances.

A Structural, Dynamic, and Thermodynamic Explanation of Thermostability in a De Novo Designed Three-Helix Bundle

UVF is a de novo designed, three-helix bundle protein built based on the backbone template of the Engrailed homeodomain (EnHD). UVF was designed in the Mayo Lab by placing only hydrophobic residues at buried positions and only polar residues at surface positions. When expressed, the protein folded as expected, and it was also highly thermostable (Tm >99°C vs. 52°C for EnHD). Hybrid proteins combining the buried and surface residue sets from UVF and EnHD were built to assess independently the contributions of UVF's fully hydrophobic core and polar surface to its thermostability. A complement of molecular dynamics simulations and equilibrium unfolding experiments were performed to quantify the proteins’ dynamics and their thermodynamic parameters of folding.

Reduction potential calculations of the Fe-S clusters in Thermus thermophilus respiratory complex I.

Respiratory complex I facilitates electron transfer from NADH to quinone over ~95 Å through a chain of seven iron-sulfur (Fe-S) clusters in the respiratory chain. In this study, the reduction potentials of the Fe-S clusters in Thermus thermophilus complex I are calculated using a Density Functional Theory + Poisson-Boltzmann method. Our results indicate that the reduction potentials are influenced by a variety of factors including the clusters being deeply buried in the complex and the protonation state of buried ionizable residues. In addition, as several of the ionizable side chains have predicted pKa values near pH 7, relatively small structural fluctuations could lead to significant (0.2 V) shifts in the reduction potential of several of the Fe-S clusters, suggesting a dynamic mechanism for electron transfer. Moreover, the method used here is a useful computational tool to study other questions about complex I. © 2019 Wiley Periodicals, Inc.

Exploring electrostatic interactions of relaxin family peptide receptor 3 and 4 with ligands using a NanoBiT-based binding assay

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely relaxin family peptide receptor 1-4 (RXFP1-4). We recently disclosed electrostatic interactions of the homologous RXFP3 and RXFP4 with some agonists based on activation complementation. However, this activation assay-based approach cannot be applied to antagonists that do not activate receptors. Herein, we propose a general approach suitable for both agonists and antagonists based on our newly-developed NanoBiT-based binding assay. We first validated the binding assay-based approach using the agonist relaxin-3, then applied it to the chimeric antagonist R3(ΔB23-27)R/I5. Three positively charged B-chain Arg residues of the agonist and antagonist were respectively replaced by a negatively charged Glu residue; meanwhile, the negatively charged Glu and Asp residue in the essential WxxExxxD motif of both receptors were respectively replaced by a positively charged Arg residue. Based on binding complementation of mutant ligands towards mutant receptors, we deduced possible electrostatic interactions of the agonist and antagonist with both RXFP3 and RXFP4: their B-chain C-terminal Arg residue interacts with the deeply buried Glu residue in the WxxExxxD motif of both receptors, and one or two of their B-chain central Arg residues interact with the shallowly buried Asp residue in the WxxExxxD motif of both receptors. Our present work shed new light on the interaction mechanism of RXFP3 and RXFP4 with agonists and antagonists, and also provided a novel approach for interaction studies of some plasma membrane receptors with their ligands.

Hyperactive mutation occurs adjacent to the essential glutamate 286 for transport in the yeast tryptophan permease Tat2

In Saccharomyces cerevisiae, high-affinity tryptophan import is mediated by the plasma membrane permease Tat2. Herein, we identified hyperactive Tat2 mutations, I285V and I285T, which allowed the cells to grow at very low tryptophan concentrations (<4 μg/mL). The Km value of wild-type Tat2 for tryptophan appeared to be 24 μg/mL, whereas that of Tat2I285V and Tat2I285T was 17 and 11 μg/mL, respectively. Normalized values of Vmax/Km for Tat2I285V- and Tat2I285T-mediated tryptophan import were 2-fold higher than that for Tat2, suggesting that these mutations increase the affinity for tryptophan, and mediate transport at very low tryptophan concentrations. I285 resides adjacent to E286, a fully conserved residue among amino acid pemreases. According to a pKa prediction for E208 (pKa ∼8.3–11.7) of Escherichia coli AdiC antiporter, a structural homologue of Tat2, the E286 carboxyl chain of Tat2 could get loaded with a proton during tryptophan/H+ symport. Hence, I285V and I285T mutations might affect the buried residue environment of Tat2, thereby facilitating tryptophan import. Additionally, Tat2I285V and Tat2I285T levels increased rapidly, and were efficiently localized to the cell surface after transferring the cells to low tryptophan medium (0.5 μg/mL). Our findings provide a clue to gain insights into the property of high-affinity transport mechanisms, and offer a unique approach to improve the functionality of broad types of amino acid permeases.

Slow Motion Protein Dance Visualized Using Red-Edge Excitation Shift of a Buried Fluorophore.

It has been extremely challenging to detect protein structures with a dynamic core, such as dry molten globules, that remain in equilibrium with the tightly packed native (N) state and that are important for a myriad of entropy-driven protein functions. Here, we detect the higher entropy conformations of a human serum protein, using red-edge excitation shift experiments. We covalently introduced a fluorophore inside the protein core and observed that in a subset of native population, the side chains of the polar and buried residues have different spatial arrangements than the mean population and that they solvate the fluorophore on a timescale much slower than the nanosecond timescale of fluorescence. Our results provide direct evidence for the dense fluidity of protein core and show that alternate side-chain packing arrangements exist in the core that might be important for multiple binding functions of this protein.

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (Tm > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.