Molecular dynamics simulations of a calmodulin-peptide complex in solution

We have performed an 4-ns MD simulation of calmodulin complexed with a target peptide in explicit water, under realistic conditions of constant temperature and pressure, in the presence of a physiological concentration of counterions and using Ewald summation to avoid truncation of long-range electrostatic forces. During the simulation the system tended to perform small fluctuations around a structure similar to, but somewhat looser than the starting crystal structure. The calmodulin-peptide complex was quite rigid and did not exhibit any large amplitude domain motions such as previously seen in apo- and calcium-bound calmodulin. We analyzed the calmodulin-peptide interactions by calculating buried surface areas, CHARMM interaction energies and continuum model interaction free energies. In the trajectory, the protein surface area buried by contact with the peptide is 1373 A(2) approximately evenly divided between the calmodulin N-terminal, C-terminal and central linker regions. A majority of this buried surface, 803 A(2), comes from nonpolar residues, in contrast to the protein as a whole, for which the surface is made up of mostly polar and charged groups. Our continuum calculations indicate that the largest favorable contribution to peptide binding comes from burial of molecular surface upon complex formation. Electrostatic contributions are favorable but smaller in the trajectory structures, and actually unfavorable for binding in the crystal structure. Since nonpolar groups make up most of buried surface of the protein, our calculations suggest that the hydrophobic effect is the main driving force for binding the helical peptide to calmodulin, consistent with thermodynamic analysis of experimental data. Besides the burial of nonpolar surface area, secondary contributions to peptide binding come from burial of polar surface and electrostatic interactions. In the nonpolar interactions a crucial role is played by the nine methionines of calmodulin. In the electrostatic interactions the negatively charged protein residues and positively charged peptide residues play a dominant role.     

Statistical analysis of protein structures suggests that buried ionizable residues in proteins are hydrogen bonded or form salt bridges

It is well known that nonpolar residues are largely buried in the interior of proteins, whereas polar and ionizable residues tend to be more localized on the protein surface where they are solvent exposed. Such a distribution of residues between surface and interior is well understood from a thermodynamic point: nonpolar side chains are excluded from the contact with the solvent water, whereas polar and ionizable groups have favorable interactions with the water and thus are preferred at the protein surface. However, there is an increasing amount of information suggesting that polar and ionizable residues do occur in the protein core, including at positions that have no known functional importance. This is inconsistent with the observations that dehydration of polar and in particular ionizable groups is very energetically unfavorable. To resolve this, we performed a detailed analysis of the distribution of fractional burial of polar and ionizable residues using a large set of ?2600 nonhomologous protein structures. We show that when ionizable residues are fully buried, the vast majority of them form hydrogen bonds and/or salt bridges with other polar/ionizable groups. This observation resolves an apparent contradiction: the energetic penalty of dehydration of polar/ionizable groups is paid off by favorable energy of hydrogen bonding and/or salt bridge formation in the protein interior. Our conclusion agrees well with the previous findings based on the continuum models for electrostatic interactions in proteins. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies

We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM-peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation.     

On the role of electrostatic interactions in the design of protein-protein interfaces

Here, the methods of continuum electrostatics are used to investigate the contribution of electrostatic interactions to the binding of four protein-protein complexes; barnase-barstar, human growth hormone and its receptor, subtype N9 influenza virus neuraminidase and the NC41 antibody, the Ras binding domain (RBD) of kinase cRaf and a Ras homologue Rap1A. In two of the four complexes electrostatics are found to strongly oppose binding (hormone-receptor and neuraminidase-antibody complexes), in one case the net effect is close to zero (barnase-barstar) and in one case electrostatics provides a significant driving force favoring binding (RBD-Rap1A). In order to help understand the wide range of electrostatic contributions that were calculated, the electrostatic free energy was partitioned into contributions of individual charged and polar residues, salt bridges and networks involving salt bridges and hydrogen bonds. Although there is no one structural feature that accounts for the differences between the four interfaces, the extent to which the desolvation of buried charges is compensated by the formation of hydrogen bonds and ion pairs appears to be an important factor. Structural features that are correlated with contribution of an individual residue to stability are also discussed. These include partial burial of a charged group in the free monomer, the formation of networks involving charged and polar amino acids, and the formation of partially exposed ion-pairs. The total electrostatic contribution to binding is found to be inversely correlated with buried total and non-polar surface area. This suggests that different interfaces can be designed to exploit electrostatic and hydrophobic forces in very different ways.     

Use of a potential of mean force to analyze free energy contributions in protein folding.

A method for calculation of the free energy of residues as a function of residue burial is proposed. The method is based on the potential of mean force, with a reaction coordinate expressed by residue burial. Residue burials are calculated from high-resolution protein structures. The largest individual contributions to the free energy of a residue are found to be due to the hydrophobic interactions of the nonpolar atoms, interactions of the main chain polar atoms, and interactions of the charged groups of residues Arg and Lys. The contribution to the free energy of folding due to the uncharged side chain polar atoms is small. The contribution to the free energy of folding due to the main chain polar atoms is favorable for partially buried residues and less favorable or unfavorable for fully buried residues. Comparison of the accessible surface areas of proteins and model spheres shows that proteins deviate considerably from a spherical shape and that the deviations increase with the size of a protein. The implications of these results for protein folding are also discussed.     

Structural evidence for a dominant role of nonpolar interactions in the binding of a transport/chemosensory receptor to its highly polar ligands.

The receptor, a maltose/maltooligosaccharide-binding protein, has been found to be an excellent system for the study of molecular recognition because its polar and nonpolar binding functions are segregated into two globular domains. The X-ray structures of the "closed" and "open" forms of the protein complexed with maltose and maltotetraitol have been determined. These sugars have approximately 3 times more accessible polar surface (from OH groups) than nonpolar surface (from small clusters of sugar ring CH bonds). In the closed structures, the oligosaccharides are buried in the groove between the two domains of the protein and bound by extensive hydrogen bonding interactions of the OH groups with the polar residues confined mostly in one domain and by nonpolar interactions of the CH clusters with four aromatic residues lodged in the other domain. Substantial contacts between the sugar hydroxyls and aromatic residues are also formed. In the open structures, the oligosaccharides are bound almost exclusively in the domain rich in aromatic residues. This finding, along with the analysis of buried surface area due to complex formations in the open and closed structures, supports a major role for nonpolar interactions in initial ligand binding even when the ligands have significantly greater potential for highly specific polar interactions.     

On the calculation of binding free energies using continuum methods: Application to MHC class I protein-peptide interactions

This paper describes a methodology to calculate the binding free energy (ΔG) of a protein-ligand complex using a continuum model of the solvent. A formal thermodynamic cycle is used to decompose the binding free energy into electrostatic and non-electrostatic contributions. In this cycle, the reactants are discharged in water, associated as purely nonpolar entities, and the final complex is then recharged. The total electrostatic free energies of the protein, the ligand, and the complex in water are calculated with the finite difference Poisson-Boltzmann (FDPB) method. The nonpolar (hydrophobic) binding free energy is calculated using a free energy-surface area relationship, with a single alkane/water surface tension coefficient (γ_aw). The loss in backbone and side-chain configurational entropy upon binding is estimated and added to the electrostatic and the nonpolar components of ΔG. The methodology is applied to the binding of the murine MHC class I protein H-2K^b with three distinct peptides, and to the human MHC class I protein HLA-A2 in complex with five different peptides. Despite significant differences in the amino acid sequences of the different peptides, the experimental binding free energy differences (ΔΔG_exp) are quite small (<0.3 and <2.7 kcal/mol for the H-2K^b and HLA-A2 complexes, respectively). For each protein, the calculations are successful in reproducing a fairly small range of values for ΔΔG_calc (<4.4 and <5.2 kcal/mol, respectively) although the relative peptide binding affinities of H-2K^b and HLA-A2 are not reproduced. For all protein-peptide complexes that were treated, it was found that electrostatic interactions oppose binding whereas nonpolar interactions drive complex formation. The two types of interactions appear to be correlated in that larger nonpolar contributions to binding are generally opposed by increased electrostatic contributions favoring dissociation. The factors that drive the binding of peptides to MHC proteins are discussed in light of our results.     

Site-Specific Peptide Probes Detect Buried Water in a Lipid Membrane

Transmembrane peptides contain polar residues in the interior of the membrane, which may alter the electrostatic environment and favor hydration in the otherwise nonpolar environment of the membrane core. Here, we demonstrate a general, nonperturbative strategy to probe hydration of the peptide backbone at specific depths within the bilayer using a combination of site-specific isotope labels, ultrafast two-dimensional infrared spectroscopy, and spectral modeling based on molecular dynamics simulations. Our results show that the amphiphilic pH-low insertion peptide supports a highly heterogeneous environment, with significant backbone hydration of nonpolar residues neighboring charged residues. For example, a leucine residue located as far as 1 nm into the hydrophobic bulk reports hydrogen-bonded populations as high as ∼20%. These findings indicate that the polar nature of these residues may facilitate the transport of water molecules into the hydrophobic core of the membrane.     

The conformation of a signal peptide bound by Escherichia coli preprotein translocase SecA

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (Kd approximately 10(-5) m). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an alpha-helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.     

Association of protein-DNA recognition complexes: electrostatic and nonelectrostatic effects

In this study the electrostatic and nonelectrostatic contributions to the binding free energy of a number of different protein-DNA recognition complexes are investigated. To determine the electrostatic effects in the protein-DNA association the Poisson-Boltzmann approach was applied. Overall the salt-dependent electrostatic free energy opposed binding in all protein-DNA complexes except one, and the salt-independent electrostatic contribution favored binding in more than half of the complexes. Further the salt-dependent electrostatic free energy increased with higher ionic concentrations and therefore complex association is stronger opposed at higher ionic concentrations. The hydrophobic effect in the protein-DNA complexes was determined from the buried accessible surface area and the surface tension. A majority of the complexes showed more polar than nonpolar buried accessible surface area. Interestingly the buried DNA-accessible surface area was preferentially hydrophilic, only in one complex a slightly more hydrophobic buried accessible surface area was observed. A quite sophisticated balance between several different free energy components seems to be responsible for determining the free energy of binding in protein-DNA systems.     

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.     

Energetics of sequence-specific protein-DNA association: computational analysis of integrase Tn916 binding to its target DNA

The N-terminal domain of the bacterial integrase Tn916 specifically recognizes the 11 bp DNA target site by positioning the face of a three-stranded beta-sheet into the major groove. Binding is linked to structural adaptation. We have characterized INT-DBD binding to DNA in detail by calorimetry [Milev, S., Gorfe, A., Karshikoff, A., Clubb, R. T., Bosshard, H. R., and Jelesarov, I. (2003) Biochemistry 42, 3481-3491]. Our thermodynamic analysis has indicated that the major driving force of association is the hydrophobic effect while polar interactions contribute less. To gain more comprehensive information about the binding process, we performed a computational analysis of the binding free energy and report here the results. A hybrid molecular mechanics/continuum approach was followed. The total binding free energy is predicted with reasonable accuracy. The calculations confirm that nonpolar effects stabilize the protein-DNA complex while electrostatics opposes binding. Structural changes optimizing surface complementarity are costly in terms of energy. The energetic consequences from the replacement of nine DNA-contacting residues by alanine were investigated. The calculations correctly predict the binding affinity decrease of eight mutations and the destabilizing effect of one wild-type residue. Bulky side chains stabilize the wild-type complex through packing interactions and favorable nonpolar dehydration, but the net nonpolar energy changes do not correlate with the relative affinity loss upon mutation. Discrete protein-DNA electrostatic interactions may be net stabilizing or net destabilizing depending on the local environment. In contrast to nonpolar energy changes, the magnitude of the electrostatic free energy ranks the mutations according to the experimentally measured DeltaDeltaG. Free energy decomposition analysis from a structural perspective leads to detailed information about the thermodynamic strategy used by INT-DBD for sequence-specific DNA binding.     

Contribution of the KCa3.1 channel–calmodulin interactions to the regulation of the KCa3.1 gating process

The Ca²⁺-activated potassium channel of intermediate conductance, KCa3.1, is now emerging as a therapeutic target for a large variety of health disorders. The Ca²⁺ sensitivity of KCa3.1 is conferred by the Ca²⁺-binding protein calmodulin (CaM), with the CaM C-lobe constitutively bound to an intracellular domain of the channel C terminus. It was proposed on the basis of the crystal structure obtained for the C-terminal region of the rat KCa2.2 channel (rSK2) with CaM that the binding of Ca²⁺ to the CaM N-lobe results in CaM interlocking the C-terminal regions of two adjacent KCa3.1 subunits, leading to the formation of a dimeric structure. A study was thus undertaken to identify residues of the CaM N-lobe–KCa3.1 complex that either contribute to the channel activation process or control the channel open probability at saturating Ca²⁺ (Pomax). A structural homology model of the KCa3.1–CaM complex was first generated using as template the crystal structure of the C-terminal region of the rat KCa2.2 channel with CaM. This model was confirmed by cross-bridging residues R362 of KCa3.1 and K75 of CaM. Patch-clamp experiments were next performed, demonstrating that the solvation energy of the residue at position 367 in KCa3.1 is a key determinant to the channel Pomax and deactivation time t_off. Mutations of residues M368 and Q364 predicted to form anchoring points for CaM binding to KCa3.1 had little impact on either t_off or Pomax. Finally, our results show that channel activation depends on electrostatic interactions involving the charged residues R362 and E363, added to a nonpolar energy contribution coming from M368. We conclude that electrostatic interactions involving residues R362 and E363 and hydrophobic effects at M368 play a prominent role in KCa3.1 activation, whereas hydrophobic interactions at S367 are determinant to the stability of the CaM–KCa3.1 complex throughout gating.     

Using Multiconformation Continuum Electrostatics to Compare Chloride Binding Motifs in α-Amylase, Human Serum Albumin, and Omp32

Ions are a ubiquitous component of the cellular environment, transferring into cells through membrane-embedded proteins. Ions bind to proteins to regulate their charge and function. Here, using multiconformation continuum electrostatics (MCCE), we show that the changes of chloride binding to α-amylase, human serum albumin (HSA) and Omp32 with pH, and of α-amylase with mutation agree well with experimental data. The three proteins represent three different types of binding. In α-amylase, chloride is bound in a specific buried site. Chloride binding is strongly coupled to the protonation state of a nearby lysine. MCCE calculates an 11-fold change in chloride affinity between the wild-type α-amylase and the K300R mutant, in good agreement with the measured 10-fold change. Without considering the coupled protonation reaction, the calculated affinity change would be more than 10⁶-fold. In HSA, chlorides are distributed on the protein surface. Although HSA has a negative net charge, it binds more anions than cations. There are no highly occupied binding sites in HSA. Rather, there are many partially occupied sites near clusters of basic residues. The relative affinity of bound ions of different charges is shown to depend on the distribution of charged residues on the surface rather than the overall net charge of the protein. The calculated strong pH dependence of the number of chlorides bound and the anion selectivity agree with those of previous experiments. In Omp32, chlorides are stabilized in an anion-selective transmembrane channel in a pH-independent manner. The positive electrostatic potential in Omp32 results in about two chlorides and no cations bound in the transmembrane region of this anion-selective channel. The studies here show that with the ability to sample multiple binding sites and coupled protein protonation states, MCCE provides a powerful tool to analyze and predict ion binding. The calculations overestimate the affinity of surface chloride in HSA and Omp32 relative to the buried ion in amylase. Differences between ion-solvent interactions for buried and surface ions will be discussed.     

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.     

Electrostatic contributions to the stability of hyperthermophilic proteins.

Electrostatic contributions to the folding free energy of several hyperthermophilic proteins and their mesophilic homologs are calculated. In all the cases studied, electrostatic interactions are more favorable in the hyperthermophilic proteins. The electrostatic free energy is found not to be correlated with the number of ionizable amino acid residues, ion pairs or ion pair networks in a protein, but rather depends on the location of these groups within the protein structure. Moreover, due to the large free energy cost associated with burying charged groups, buried ion pairs are found to be destabilizing unless they undergo favorable interactions with additional polar groups, including other ion pairs. The latter case involves the formation of stabilizing ion pair networks as is observed in a number of proteins. Ion pairs located on the protein surface also provide stabilizing interactions in a number of cases. Taken together, our results suggest that many hyperthermophilic proteins enhance electrostatic interactions through the optimum placement of charged amino acid residues within the protein structure, although different design strategies are used in different cases. Other physical mechanisms are also likely to contribute, however optimizing electrostatic interactions offers a simple means of enhancing stability without disrupting the core residues characteristic of different protein families.     

Peptide-membrane interactions A fluorescence study of the binding of oligopeptides containing aromatic and basic residues to phospholipid vesicles

The binding of oligopeptides containing basic and aromatic residues to phospholipid vesicles has been studied by fluorescence spectroscopy. Tryptophan-containing peptide such as Lys-Trp-Lys or Lys-Trp(OMe) exhibit a shift of their fluorescence toward shorter wavelengths and an increased fluorescence quantum yield upon binding to phosphatidylinositol (PI) or phosphatidylserine (PS) vesicles. No binding was detected with phosphatidylcholine vesicles. The binding is strongly dependent on ionic strength and pH. Binding decreases when ionic strength increases indicating an important role of electrostatic interactions. The pH-dependence of binding reveals that the apparent $\text{p}\text{K}$ of the terminal carboxyl group of Lys-Trp-Lys is raised by ～3 units upon binding to PI and PS vesicles. The binding of tyrosine-containing peptides to PI and PS vesicles is characterized by an increase in the fluorescence quantum yield of the peptide without any shift in fluorescence maximum. A natural nonapeptide from the myelin basic protein which contains one tryptophan residue binds to PI and PS vesicles at low pH when the acidic groups are neutralized. This binding is accompanied by a shift of the tryptophyl fluorescence toward shorter wavelengths together with an enhancement of the fluorescence quantum yield. Dissociation of the complex is achieved at high ionic strength. These results indicate that aromatic residues of oligopeptides bound to the phospholipid polar heads by electrostatic interactions become buried in a more hydrophobic environment in the vicinity of the aliphatic chains of the lipids.     

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 (T_m > 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.