Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

Partially folded equilibrium intermediate of the villin headpiece HP67 defined by 13C relaxation dispersion

Identification and characterization of ensembles of intermediate states remains an important objective in describing protein folding in atomic detail. The 67-residue villin headpiece, HP67, consists of an N-terminal subdomain (residues 10–42) that transiently unfolds at equilibrium under native-like conditions and a highly stable C-terminal subdomain (residues 43–76). The transition between folded and unfolded states of the N-terminal domain has been characterized previously by ¹⁵N NMR relaxation dispersion measurements (Grey et al. in J Mol Biol 355:1078, 2006). In the present work, ¹³C spin relaxation was used to further characterize backbone and hydrophobic core contributions to the unfolding process. Relaxation of ¹³C^α spins was measured using the Hahn echo technique at five static magnetic fields (11.7, 14.1, 16.4, 18.8, and 21.1 T) and the Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion method at a static magnetic field of 14.1 T. Relaxation of methyl ¹³C spins was measured using CPMG relaxation dispersion experiments at static magnetic fields of 14.1 and 18.8 T. Results for ¹³C and ¹⁵N spins yielded a consistent model in which the partially unfolded intermediate state of the N-terminal subdomain maintains residual structure for residues near the unprotonated His41 imidazole ring and in the interface between the N- and C-terminal subdomains. In addition, a second faster process was detected that appears to represent local dynamics within the folded state of the molecule and is largely confined to the hydrophobic interface between the N- and C-terminal subdomains.     

NMR structure of an F-actin-binding "headpiece" motif from villin

A growing family of F-actin-bundling proteins harbors a modular F-actin-binding headpiece domain at the C terminus. Headpiece provides one of the two F-actin-binding sites essential for filament bundling. Here, we report the first structure of a functional headpiece domain. The NMR structure of chicken villin headpiece (HP67) reveals two subdomains that share a tightly packed hydrophobic core. The N-terminal subdomain contains bends, turns, and a four-residue alpha-helix as well as a buried histidine residue that imparts a pH-dependent folding. The C-terminal subdomain is composed of three alpha-helices and its folding is pH-independent. Two residues previously implicated in F-actin-binding form a buried salt-bridge between the N and C-terminal subdomains. The rest of the identified actin-binding residues are solvent-exposed and map onto a unique F-actin-binding surface.     

On the unyielding hydrophobic core of villin headpiece

Villin headpiece (HP67) is a small, autonomously-folding domain that has become a model system for understanding the fundamental tenets governing protein folding. In this communication, we explore the role that Leu61 plays in the structure and stability of the construct. Deletion of Leu61 results in a completely unfolded protein that cannot be expressed in Escherichia coli. Omission of only the aliphatic leucine side chain (HP67 L61G) perturbed neither the backbone conformation nor the orientation of local hydrophobic side chains. As a result, a large, solvent-exposed hydrophobic pocket, a negative replica of the leucine side-chain, was created on the surface. The loss of the hydrophobic interface between leucine 61 and the hydrophobic pocket destabilized the construct by ~3.3 kcal/mol. Insertion of a single glycine residue immediately before Leu61 (HP67 L61[GL]) was also highly destabilizing and had the effect of altering the backbone conformation (α-helix to π-helix) in order to precisely preserve the wild-type position and conformation of all hydrophobic residues, including Leu61. In addition to demonstrating that the hydrophobic side-chain of Leu61 is critically important for the stability of villin headpiece, our results are consistent with the notion that the precise interactions present within the hydrophobic core, rather than the hydrogen bonds that define the secondary structure, specify a protein's fold.     

Folding stability modulation of the villin headpiece helical subdomain by 4‐fluorophenylalanine and 4‐methylphenylalanine

HP36, the helical subdomain of villin headpiece, contains a hydrophobic core composed of three phenylalanine residues (Phe47, Phe51, and Phe58). Hydrophobic effects and electrostatic interactions were shown to be the critical factors in stabilizing this core and the global structure. To assess the interactions among Phe47, Phe51, and Phe58 residues and investigate how they affect the folding stability, we implanted 4‐fluorophenylalanine (Z) and 4‐methylphenylalanine (X) into the hydrophobic core of HP36. We chemically synthesized HP36 and its seven variants including four single mutants whose Phe51 or Phe58 was replaced with Z or X, and three double mutants whose Phe51 and Phe58 were both substituted. Circular dichroism and nuclear magnetic resonance measurements show that the variants exhibit a native HP36 like fold, of which F51Z and three double mutants are more stable than the wild type. Molecular modeling provided detailed interaction energy within the phenylalanine residues, revealing that electrostatic interactions dominate the stability modulation upon the introduction of 4‐fluorophenylalanine and 4‐methylphenylalanine. Our results show that these two non‐natural amino acids can successfully tune the interactions in a relatively compact hydrophobic core and the folding stability without inducing dramatic steric effects. Such an approach may be applied to other folded motifs or proteins. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 627–637, 2015.     

Slow motions in the hydrophobic core of chicken villin headpiece subdomain and their contributions to configurational entropy and heat capacity from solid-state deuteron NMR measurements

We have investigated microsecond to millisecond time scale dynamics in several key hydrophobic core methyl groups of chicken villin headpiece subdomain protein (HP36) using a combination of single-site labeling, deuteron solid-state NMR line shape analysis, and computational modeling. Deuteron line shapes of hydrated powder samples are dominated by rotameric jumps and show a large variability of rate constants, activation energies, and rotameric populations. Site-specific activation energies vary from 6 to 38 kJ/mol. An additional mode of diffusion on a restricted arc is significant for some sites. In dry samples, the dynamics is quenched. Parameters of the motional models allow for calculations of configurational entropy and heat capacity, which, together with the rate constants, allow for observation of interplay between thermodynamic and kinetic picture of the landscape. Mutations at key phenylalanine residues at both distal (F47L&F51L) and proximal (F58L) locations to a relatively rigid side chain of L69 have a pronounced effect on alleviating the rigidity of this side chain at room temperature and demonstrate the sensitivity of the hydrophobic core environment to such perturbations.     

Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.     

Multistate folding of the villin headpiece domain

The villin headpiece (HP67) is a 67 residue, monomeric protein derived from the C-terminal domain of villin. Wild-type HP67 (WT HP67) is the smallest fragment of villin that retains strong in vitro actin-binding activity. WT HP67 is made up of two subdomains, which form a tightly packed interface. The C-terminal subdomain of WT HP67, denoted HP35, is rich in helical structure, folds in isolation, and has been widely used as a model system for folding studies. In contrast, very little is known about the folding of the intact villin headpiece domain. Here, NMR, CD and H/2H amide exchange measurements are used to follow the pH, thermal and urea-induced unfolding of WT HP67 and a mutant (HP67 H41Y) in which a buried conserved histidine in the N-terminal subdomain, His41, has been mutated to Tyr. Although most small proteins display two-state equilibrium unfolding, the results presented here demonstrate that unfolding of the villin headpiece is a multistate process. The presence of a folded N-terminal subdomain is shown to stabilize the C-terminal subdomain, increasing the midpoints of the thermal and urea-induced unfolding transitions and increasing protection factors for H/2H exchange. Histidine 41 has been shown to act as a pH-dependent switch in wild-type HP67: the N-terminal subdomain is unfolded when His41 is protonated, while the C-terminal subdomain remains folded irrespective of the protonation state of His41. Mutation of His41 to Tyr eliminates the segmental pH-dependent unfolding of the headpiece. The mutation stabilizes both domains, but folding is still multistate, indicating that His41 is not solely responsible for the unusual equilibrium unfolding behavior of villin headpiece domain.     

Salt effects on hydrophobic‐core formation in folding of a helical miniprotein studied by molecular dynamics simulations

We have investigated effects of salt ions on folding events of a helical miniprotein chicken villin headpiece subdomain HP36. Low concentrations of ions alter electrostatic interactions between charged groups of a protein and can change the populations of conformers. Here, we compare two data sets of folding simulations of HP36 in explicit water solvent with or without ions. For efficient sampling of the conformational space of HP36, the multicanonical replica‐exchange molecular dynamics method was employed. Our analyses suggest that salt alters salt‐bridging nature of the protein at later stages of folding at room temperature. Especially, more nonnative, nonlocal salt bridges are formed at near‐native conformations in pure water. Our analyses also show that such salt‐bridge formation hinders the fully native hydrophobic‐core packing at the final stages of folding. Proteins 2014; 82:933–943. © 2013 Wiley Periodicals, Inc.     

The unusual internal motion of the villin headpiece subdomain

The thermostable 36‐residue subdomain of the villin headpiece (HP36) is the smallest known cooperatively folding protein. Although the folding and internal dynamics of HP36 and close variants have been extensively studied, there has not been a comprehensive investigation of side‐chain motion in this protein. Here, the fast motion of methyl‐bearing amino acid side chains is explored over a range of temperatures using site‐resolved solution nuclear magnetic resonance deuterium relaxation. The squared generalized order parameters of methyl groups extensively spatially segregate according to motional classes. This has not been observed before in any protein studied using this methodology. The class segregation is preserved from 275 to 305 K. Motions detected in Helix 3 suggest a fast timescale of conformational heterogeneity that has not been previously observed but is consistent with a range of folding and dynamics studies. Finally, a comparison between the order parameters in solution with previous results based on solid‐state nuclear magnetic resonance deuterium line shape analysis of HP36 in partially hydrated powders shows a clear disagreement for half of the sites. This result has significant implications for the interpretation of data derived from a variety of approaches that rely on partially hydrated protein samples.     

Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain

The contribution of interactions involving the imidazole ring of His41 to the pH-dependent stability of the villin headpiece (HP67) N-terminal subdomain has been investigated by nuclear magnetic resonance (NMR) spin relaxation. NMR-derived backbone N-H order parameters (S2) for wild-type (WT) HP67 and H41Y HP67 indicate that reduced conformational flexibility of the N-terminal subdomain in WT HP67 is due to intramolecular interactions with the His41 imidazole ring. These interactions, together with desolvation effects, contribute to significantly depress the pKa of the buried imidazole ring in the native state. 15N R1rho relaxation dispersion data indicate that WT HP67 populates a partially folded intermediate state that is 10.9 kJ mol(-1) higher in free energy than the native state under non-denaturing conditions at neutral pH. The partially folded intermediate is characterized as having an unfolded N-terminal subdomain while the C-terminal subdomain retains a native-like fold. Although the majority of the residues in the N-terminal subdomain sample a random-coil distribution of conformations, deviations of backbone amide 1H and 15N chemical shifts from canonical random-coil values for residues within 5A of the His41 imidazole ring indicate that a significant degree of residual structure is maintained in the partially folded ensemble. The pH-dependence of exchange broadening is consistent with a linear three-state exchange model whereby unfolding of the N-terminal subdomain is coupled to titration of His41 in the partially folded intermediate with a pKa,I=5.69+/-0.07. Although maintenance of residual interactions with the imidazole ring in the unfolded N-terminal subdomain appears to reduce pKa,I compared to model histidine compounds, protonation of His41 disrupts these interactions and reduces the difference in free energy between the native state and partially folded intermediate under acidic conditions. In addition, chemical shift changes for residues Lys70-Phe76 in the C-terminal subdomain suggest that the HP67 actin binding site is disrupted upon unfolding of the N-terminal subdomain, providing a potential mechanism for regulating the villin-dependent bundling of actin filaments.     

Intrinsic dynamics of the partly unstructured PX domain from the Sendai virus RNA polymerase cofactor P

Despite their evident importance for function, dynamics of intrinsically unstructured proteins are poorly understood. Sendai virus phosphoprotein, cofactor of the RNA polymerase, contains a partly unstructured protein domain. The phosphoprotein X domain (PX) is responsible for binding the polymerase to the nucleocapsid assembling the viral RNA. For RNA synthesis, the interplay of the dynamics of the unstructured and structured PX subdomains is thought to drive progression of the RNA polymerase along the nucleocapsid. Here we present a detailed study of the dynamics of PX using hydrogen/deuterium exchange and different NMR relaxation measurements. In the unstructured subdomain, large amplitude fast motions were found to be fine-tuned by the presence of residues with short side chains. In the structured subdomain, where fast motions of both backbone and side chains are fairly restricted, the first helix undergoes slow conformational exchange corresponding to a local unfolding event. The other two helices, which represent the nucleocapsid binding site, were found to be more stable and to reorient with respect to each other, as probed by slow conformational exchange identified for residues on the third helix. The study illustrates the intrinsically differential dynamics of this partly unstructured protein and proposes the relation between these dynamics and its function.     

Hydrophobic Core Formation and Dehydration in Protein Folding Studied by Generalized-Ensemble Simulations

Despite its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed at least five events of HP36 folding into nativelike conformations. The smallest backbone root mean-square deviation from the crystal structure was 1.1 Å. In the nativelike conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories show the following sequential events in folding of HP36: 1), Helix 3 is formed at the earliest stage; 2), the backbone and the side chains near the loop between Helices 2 and 3 take nativelike conformations; and 3), the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. This sequence suggests that the initial folding nucleus is not necessarily the same as the hydrophobic core, consistent with a recent experimental ϕ-value analysis.     

Deuteration of nonexchangeable protons on proteins affects their thermal stability,side‐chain dynamics,and hydrophobicity

We have investigated the effect of deuteration of non‐exchangeable protons on protein global thermal stability, hydrophobicity, and local flexibility using well‐known thermostable model systems such as the villin headpiece subdomain (HP36) and the third immunoglobulin G‐binding domain of protein G (GB3). Reversed‐phase high‐performance liquid chromatography (RP‐HPLC) measurements as a function of temperature probe global thermal stability in the presence of acetonitrile, while differential scanning calorimetry determines thermal stability in solution. Both indicate small but measurable changes in the order of several degrees. RP‐HPLC also permitted quantification of the effect of deuteration of just three core phenylalanine side chains of HP36. NMR dynamics investigation has focused on methyl axes motions using cross‐correlated relaxation measurements. The analysis of order parameters provided a complex picture indicating that deuteration generally increases motional amplitudes of sub‐nanosecond motion in GB3 but decreases those in HP36. Combined with earlier dynamics measurements at C_α–C_β sites and backbone sites of GB3, which probed slower time scales, the results point to the need to probe multiple atoms in the protein and variety of time scales to the discern the full complexity of the effects of deuteration on dynamics.     

Sequential unfolding of the hemolysin two‐partner secretion domain from Proteus mirabilis

Protein secretion is a major contributor to Gram‐negative bacterial virulence. Type Vb or two‐partner secretion (TPS) pathways utilize a membrane bound β‐barrel B component (TpsB) to translocate large and predominantly virulent exoproteins (TpsA) through a nucleotide independent mechanism. We focused our studies on a truncated TpsA member termed hemolysin A (HpmA265), a structurally and functionally characterized TPS domain from Proteus mirabilis. Contrary to the expectation that the TPS domain of HpmA265 would denature in a single cooperative transition, we found that the unfolding follows a sequential model with three distinct transitions linking four states. The solvent inaccessible core of HpmA265 can be divided into two different regions. The C‐proximal region contains nonpolar residues and forms a prototypical hydrophobic core as found in globular proteins. The N‐proximal region of the solvent inaccessible core, however, contains polar residues. To understand the contributions of the hydrophobic and polar interiors to overall TPS domain stability, we conducted unfolding studies on HpmA265 and site‐specific mutants of HpmA265. By correlating the effect of individual site‐specific mutations with the sequential unfolding results we were able to divide the HpmA265 TPS domain into polar core, nonpolar core, and C‐terminal subdomains. Moreover, the unfolding studies provide quantitative evidence that the folding free energy for the polar core subdomain is more favorable than for the nonpolar core and C‐terminal subdomains. This study implicates the hydrogen bonds shared among these conserved internal residues as a primary means for stabilizing the N‐proximal polar core subdomain.     

Conformational changes of FtsZ reported by tryptophan mutants

E. coli FtsZ has no native tryptophan. We showed previously that the mutant FtsZ L68W gave a 2.5-fold increase in trp fluorescence when assembly was induced by GTP. L68 is probably buried in the protofilament interface upon assembly, causing the fluorescence increase. In the present study we introduced trp residues at several other locations and examined them for assembly-induced fluorescence changes. L189W, located on helix H7 and buried between the N- and C-terminal subdomains, showed a large fluorescence increase, comparable to L68W. This may reflect a shift or rotation of the two subdomains relative to each other. L160W showed a smaller increase in fluorescence, and Y222W a decrease in fluorescence, upon assembly. These two are located on the surface of the N and C subdomains, near the domain boundary. The changes in fluorescence may reflect movements of the domains or of nearby side chains. We prepared a double mutant Y222W/S151C and coupled ATTO-655 to the cys. The Cα of trp in the C-terminal subdomain was 10 ? away from that of the cys in the N-terminal subdomain, permitting the ATTO to make van der Waals contact with the trp. The ATTO fluorescence showed strong tryptophan-induced quenching. The quenching was reduced following assembly, consistent with a movement apart of the two subdomains. Movements of one to several angstroms are probably sufficient to account for the changes in trp fluorescence and trp-induced quenching of ATTO. Assembly in GDP plus DEAE dextran produces tubular polymers that are related to the highly curved, mini-ring conformation. No change in trp fluorescence was observed upon assembly of these tubes, suggesting that the mini-ring conformation is the same as that of a relaxed, monomeric FtsZ.     

Mapping the cofilin binding site on yeast G-actin by chemical cross-linking

Cofilin is a major cytoskeletal protein that binds to both monomeric actin (G-actin) and polymeric actin (F-actin) and is involved in microfilament dynamics. Although an atomic structure of the G-actin-cofilin complex does not exist, models of the complex have been built using molecular dynamics simulations, structural homology considerations, and synchrotron radiolytic footprinting data. The hydrophobic cleft between actin subdomains 1 and 3 and, alternatively, the cleft between actin subdomains 1 and 2 have been proposed as possible high-affinity cofilin binding sites. In this study, the proposed binding of cofilin to the subdomain 1/subdomain 3 region on G-actin has been probed using site-directed mutagenesis, fluorescence labeling, and chemical cross-linking, with yeast actin mutants containing single reactive cysteines in the actin hydrophobic cleft and with cofilin mutants carrying reactive cysteines in the regions predicted to bind to G-actin. Mass spectrometry analysis of the cross-linked complex revealed that cysteine 345 in subdomain 1 of mutant G-actin was cross-linked to native cysteine 62 on cofilin. A cofilin mutant that carried a cysteine substitution in the α3-helix (residue 95) formed a cross-link with residue 144 in actin subdomain 3. Distance constraints imposed by these cross-links provide experimental evidence for cofilin binding between actin subdomains 1 and 3 and fit a corresponding docking-based structure of the complex. The cross-linking of the N-terminal region of recombinant yeast cofilin to actin residues 346 and 374 with dithio-bis-maleimidoethane (12.4 Å) and via disulfide bond formation was also documented. This set of cross-linking data confirms the important role of the N-terminal segment of cofilin in interactions with G-actin.     

Exploring the effect of silicene monolayer on the structure and function of villin headpiece and amyloid fibrils by molecular dynamics simulations

With the development of various nanomaterial expected to be used in biomedical fields, it is more important to evaluate and understand their potential effects on biological system. In this work, two proteins with different structure, Villin Headpiece (HP35) with α‐helix structure and protofibrils Aβ1‐42 with five β‐strand chains, were selected and their interactions with silicene were studied by means of molecular dynamics (MD) simulation to reveal the potential effect of silicene on the structure and function of biomolecules. The obtained results indicated that silicene could rapidly attract HP35 and Aβ1‐42 fibrils onto the surface to form a stable binding. The adsorption strength was moderate and no significant structural distortion of HP35 and Aβ1‐42 fibrils was observed. Moreover, the strength of calculated the H‐bonds in neighbor chain of Aβ1‐42 fibrils indicated that the mild interactions between silicene and fibrils could regularize the structure of Aβ1‐42 fibrils and stabilize the interactions between five chains of fibrils protein, which might enhance the aggregation of Aβ1‐42 fibrils. This study provides a new insight for understanding the interaction between nanomaterials and biomolecules and moves forward the development of silicene into biomedical fields.     

Structure and dynamics of the fatty acid binding cavity in apo rat intestinal fatty acid binding protein.

The structure and dynamics of the fatty acid binding cavity in I-FABP (rat intestinal fatty acid binding protein) were analyzed. In the crystal structure of apo I-FABP, the probe occupied cavity volume and surface are 539+/-8 A3 and 428 A2, respectively (1.4 A probe). A total of 31 residues contact the cavity with their side chains. The side-chain cavity surface is partitioned according to the residue type as follows: 36-39% hydrophobic, 21-25% hydrophilic, and 37-43% neutral or ambivalent. Thus, the cavity surface is neither like a typical protein interior core, nor is like a typical protein external surface. All hydrophilic residues that contact the cavity-with the exception of Asp74-are clustered on the one side of the cavity. The cavity appears to expand its hydrophobic surface upon fatty acid binding on the side opposite to this hydrophilic patch. In holo I-FABP the fatty acid chain interactions with the hydrophilic side chains are mediated by water molecules. Molecular dynamics (MD) simulation of fully solvated apo I-FABP showed global conformational changes of I-FABP, which resulted in a large, but seemingly transient, exposure of the cavity to the external solvent. The packing density of the side chains lining the cavity, studied by Voronoi volumes, showed the presence of two distinctive small hydrophobic cores. The MD simulation predicts significant structural perturbations of the cavity on the subnanosecond time scale, which are capable of facilitating exchange of I-FABP internal water.