Temperature-dependent dynamics of the villin headpiece helical subdomain,an unusually small thermostable protein

(15)N spin relaxation experiments were used to measure the temperature-dependence of protein backbone conformational fluctuations in the thermostable helical subdomain, HP36, of the F-actin-binding headpiece domain of chicken villin. HP36 is the smallest domain of a naturally occurring protein that folds cooperatively to a compact native state. Spin-lattice, spin-spin, and heteronuclear nuclear Overhauser effect relaxation data for backbone amide (15)N spins were collected at five temperatures in the range of 275-305 K. The data were analyzed using a model-free formalism to determine generalized order parameters, S, that describe the distribution of N-H bond vector orientations in a molecular reference frame. A novel parameter, Lambda=dln(1-S)/dln T is introduced to characterize the temperature-dependence of S. An average value of Lambda=4.5 is obtained for residues in helical conformations in HP36. This value of Lambda is not reproduced by model potential energy functions commonly used to parameterize S. The maximum entropy principle was used to derive a new model potential function that reproduces both S and Lambda. Contributions to the entropy, S(r), and heat capacity, C(r)(p), from reorientational conformational fluctuations were analyzed using this potential energy function. Values of S(r) show a qualitative dependence on S similar to that obtained for the diffusion-in-a-cone model; however, quantitative differences of up to 0.5k, in which k is the Boltzmann constant, are observed. Values of C(r)(p) approach zero for small values of S and approach k for large values of S; the largest values of C(r)(p) are predicted to occur for intermediate values of S. The results suggest that backbone dynamics, as probed by relaxation measurements, make very little contribution to the heat capacity difference between folded and unfolded states for HP36.     

Rational design, structural and thermodynamic characterization of a hyperstable variant of the villin headpiece helical subdomain

A hyperstable variant of the small independently folded helical subdomain (HP36) derived from the F-actin binding villin headpiece was designed by targeting surface electrostatic interactions and helical propensity. A double mutant N68A, K70M was significantly more stable than wild type. The Tm of wild type in aqueous buffer is 73.0 degrees C, whereas the double mutant did not display a complete unfolding transition. The double mutant could not be completely unfolded even by 10 M urea. In 3 M urea, the Tm of wild type is 54.8 degrees C while that of the N68AK70M double mutant is 73.9 degrees C. Amide H/2H exchange studies show that the pattern of exchange is very similar for wild type and the double mutant. The structures of a K70M single mutant and the double mutant were determined by X-ray crystallography and are identical to that of the wild type. Analytical ultracentrifugation demonstrates that the proteins are monomeric. The hyperstable mutant described here is expected to be useful for folding studies of HP36 because studies of the wild type domain have sometimes been limited by its marginal stability. The results provide direct evidence that naturally occurring miniature protein domains have not been evolutionarily optimized for global stability. The stabilizing effect of this double mutant could not be predicted by sequence analysis because K70 is conserved in the larger intact headpiece for functional reasons.     

The unfolded state of the villin headpiece helical subdomain: computational studies of the role of locally stabilized structure

The 36 residue villin headpiece helical subdomain (HP36) is one of the fastest cooperatively folding proteins, folding on the microsecond timescale. HP36's simple three helix topology, fast folding and small size have made it an attractive model system for computational and experimental studies of protein folding. Recent experimental studies have explored the denatured state of HP36 using fragment analysis coupled with relatively low-resolution spectroscopic techniques. These studies have shown that there is apparently only a small tendency to form locally stabilized secondary structure. Here, we complement the experimental studies by using replica exchange molecular dynamics with explicit solvent to investigate the structural features of these peptide models of unfolded HP36. To ensure convergence, two sets of simulations for each fragment were performed with different initial structures, and simulations were continued until these generated very similar final ensembles. These simulations reveal low populations of native-like structure and early folding events that cannot be resolved by experiment. For each fragment, calculated J-coupling constants and helical propensities are in good agreement with experimental trends. HP-1, corresponding to residues 41 to 53 and including the first alpha-helix, contains the highest helical population. HP-3, corresponding to residues 62 through 75 and including the third alpha-helix, contains a small population of helical turn residing at the N terminus while HP-2, corresponding to residues 52 through 61 and including the second alpha-helix, formed little to no structure in isolation. Overall, HP-1 was the only fragment to adopt a native-like conformation, but the low population suggests that formation of significant structure only occurs after formation of specific tertiary interactions.     

Effect of subdomain interactions on methyl group dynamics in the hydrophobic core of villin headpiece protein

Thermostable villin headpiece protein (HP67) consists of the N‐terminal subdomain (residues 10–41) and the autonomously folding C‐terminal subdomain (residues 42–76) which pack against each other to form a structure with a unified hydrophobic core. The X‐ray structures of the isolated C‐terminal subdomain (HP36) and its counterpart in HP67 are very similar for the hydrophobic core residues. However, fine rearrangements of the free energy landscape are expected to occur because of the interactions between the two subdomains. We detect and characterize these changes by comparing the µs‐ms time scale dynamics of the methyl‐bearing side chains in isolated HP36 and in HP67. Specifically, we probe three hydrophobic side chains at the interface of the two subdomains (L42, V50, and L75) as well as at two residues far from the interface (L61 and L69). Solid‐state deuteron NMR techniques are combined with computational modeling for the detailed characterization of motional modes in terms of their kinetic and thermodynamic parameters. The effect of interdomain interactions on side chain dynamics is seen for all residues but L75. Thus, changes in dynamics because of subdomain interactions are not confined to the site of perturbation. One of the main results is a two‐ to threefold increase in the value of the activation energies for the rotameric mode of motions in HP67 compared with HP36. Detailed analysis of configurational entropies and heat capacities complement the kinetic view of the degree of the disorder in the folded state.     

NMR characterization of a peptide model provides evidence for significant structure in the unfolded state of the villin headpiece helical subdomain

The villin headpiece subdomain (HP36) is the smallest naturally occurring protein that folds cooperatively. The protein folds on a microsecond time scale. Its small size and very rapid folding have made it a popular target for biophysical studies of protein folding. Temperature-dependent one-dimensional (1D) NMR studies of the full-length protein together with CD and 1D NMR studies of the 21-residue peptide fragment (HP21) derived from HP36 have shown that there is significant structure in the unfolded state of HP36 and have demonstrated that HP21 is a good model of these interactions. Here, we characterized the model peptide HP21 in detail by two-dimensional NMR. Strongly upfield shifted C(alpha) protons, the magnitude of the 3J(NH,alpha) coupling constants, and the pattern of backbone-backbone and backbone-side chain NOEs indicate that the ensemble of structures populated by HP21 contains alpha-helical structure and native as well as non-native hydrophobic contacts. The hydrogen-bonded secondary structure inferred from the NOEs is, however, not sufficient to confer significant protection against amide H-D exchange. These studies indicate that there is significant secondary structure and hydrophobic clustering in the unfolded state of HP36. The implications for the folding of HP36 are discussed.     

Folding of the villin headpiece subdomain from random structures. Analysis of the charge distribution as a function of pH

The structure of the 36 residue villin headpiece subdomain is investigated with the electrostatically driven Monte Carlo method. The ECEPP/3 (Empirical Conformational Energy Program for Peptides) force field, plus two different continuum solvation models, were used to describe the conformational energy of the chain with both blocked and unblocked N and C termini. A statistical analysis of an ensemble of ab initio generated conformations was carried out, based on a comparison with a set of ten native-like structures derived from published experimental data, by using rigid geometry and NMR-derived constraints obtained at pH 3.7. The ten native-like structures satisfy the NMR-derived constraints. The whole ensemble of conformations of the terminally unblocked villin headpiece sub-domain, generated by using ECEPP/3 with a continuum solvation model, were subsequently evaluated at pH 3.7 with a potential function that includes ECEPP/3 combined with a fast multigrid boundary element method. At pH 3.7, the lowest-energy conformation found during the conformational search satisfies approximately 70% of both the distance and the dihedral-angle constraints, and possesses the characteristic packing of three phenylalanine residues that constitute the main part of the hydrophobic core of the molecule. On the other hand, computations at pH 3.7 and pH 7.0 for the ten native-like structures satisfying the NMR-derived constraints indicate a substantial change in the charge distribution for each type of amino acid residue with the change in pH. The results of this study provide a basis to understand the effect of the interactions, such as hydrophobicity, charge-charge interaction and solvent polarization, on the stability of this small alpha-helical protein.     

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.     

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.     

Study of early events in the protein folding of villin headpiece using molecular dynamics simulation

Protein folding is scientifically and computationally challenging problem. The early phases of protein folding are interesting due to various events like nascent secondary structure formation, hydrophobic collapse leading to formation of non-native or meta-stable conformations. These events occur within a very short time span of 100ns as compared to total folding time of few microseconds. It is highly difficult to observe these events experimentally due to very short lifetime. Molecular dynamics simulation technique can efficiently probe the detailed atomic level understanding about these events. In the present paper, all atom molecular dynamics simulation trajectory of nearly 200ns was carried out for fully solvated villin headpiece with PME treatment using AMBER 7 package. Initial hydrophobic collapse along with secondary structure formation resulted into formation of partially stable non-native conformations. The formation of secondary structural elements and hydrophobic collapse takes place simultaneously in the folding process.     

Efficient high level expression of peptides and proteins as fusion proteins with the N-terminal domain of L9: application to the villin headpiece helical subdomain

The efficient expression of small to midsize polypeptides and small marginally stable proteins can be difficult. A new protein fusion system is developed to allow the expression of peptides and small proteins. The polypeptide of interest is linked via a Factor Xa cleavage sequence to the C-terminus of the N-terminal domain of the ribosomal protein L9 (NTL9). NTL9 is a small (56 residue) basic protein. The C-terminus of the protein is part of an alpha-helix which extends away from the globular structure thus additional domains can be fused without altering the fold of NTL9. NTL9 expresses at high levels, is extremely soluble, and remains fully folded over a wide temperature and pH range. The protein has a high net positive charge, facilitating purification of fusion proteins by ion exchange chromatography. NTL9 fusions can also be easily purified by reverse phase HPLC. As a test case we demonstrate the high level expression of a small, 36 residue, three helix bundle, the villin headpiece subdomain. This protein is widely used as a model system for folding studies and the development of a simple expression system should facilitate experimental studies of the subdomain. The yield of purified fusion protein is 70 mg/L of culture and the yield of purified villin headpiece subdomain is 24 mg/L of culture. We also demonstrate the use of the fusion system to express a smaller marginally folded peptide fragment of the villin headpiece domain.     

Heterogeneity even at the speed limit of folding: large-scale molecular dynamics study of a fast-folding variant of the villin headpiece

We have performed molecular dynamics simulations on a set of nine unfolded conformations of the fastest-folding protein yet discovered, a variant of the villin headpiece subdomain (HP-35 NleNle). The simulations were generated using a new distributed computing method, yielding hundreds of trajectories each on a time scale comparable to the experimental folding time, despite the large (10,000 atom) size of the simulation system. This strategy eliminates the need to assume a two-state kinetic model or to build a Markov state model. The relaxation to the folded state at 300 K from the unfolded configurations (generated by simulation at 373 K) was monitored by a method intended to reflect the experimental observable (quenching of tryptophan by histidine). We also monitored the relaxation to the native state by directly comparing structural snapshots with the native state. The rate of relaxation to the native state and the number of resolvable kinetic time scales both depend upon starting structure. Moreover, starting structures with folding rates most similar to experiment show some native-like structure in the N-terminal helix (helix 1) and the phenylalanine residues constituting the hydrophobic core, suggesting that these elements may exist in the experimentally relevant unfolded state. Our large-scale simulation data reveal kinetic complexity not resolved in the experimental data. Based on these findings, we propose additional experiments to further probe the kinetics of villin folding.     

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69-74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.     

Peptide models provide evidence for significant structure in the denatured state of a rapidly folding protein: the villin headpiece subdomain

The villin headpiece subdomain is a cooperatively folded 36-residue, three-alpha-helix protein. The domain is one of the smallest naturally occurring sequences which has been shown to fold. Recent experimental studies have shown that it folds on the 10-micros time scale. Its small size, simple topology, and very rapid folding have made it an attractive target for computational studies of protein folding. We present temperature-dependent NMR studies that provide evidence for significant structure in the denatured state of the headpiece subdomain. A set of peptide fragments derived from the headpiece were also characterized in order to determine if there is a significant tendency to form a locally stabilized structure in the denatured state. Peptides corresponding to each of the three isolated helices and to the connection between the first and second helices were largely unstructured. A longer peptide fragment which contains the first and second helices shows considerable structure, as judged by NMR and CD. Concentration-dependent CD measurements and analytical ultracentrifugation experiments indicate that the structure is not due to self-association. NMR studies indicate that the structure is stabilized by tertiary interactions involving phenylalanines and Val 50. A peptide in which two of the three phenylalanines are changed to leucine is considerably less structured, confirming the importance of the phenylalanines. This work indicates that there is significant structure in the denatured state of this rapidly folding protein.     

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution

The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, and chemically induced dynamic nuclear polarization (CIDNP) data.     

Temperature-dependent molecular dynamics and restrained X-ray refinement simulations of a Z-DNA hexamer

Molecular dynamics simulations of the Z-DNA hexamer 5BrdC-dG-5BrdC-dG-5BrdC-dG were performed at several temperatures between 100 K and 300 K. Above 250 K, a strong sequence-dependent flexibility in the nucleic acid is observed, with the guanine sugar and the phosphate of GpC sequences much more mobile than the cytosine sugar and phosphate of CpG sequences. At 300 K, the hexamer is in dynamic equilibrium between several Z forms, including the crystallographically determined ZI and ZII forms. The local base-pair geometry, however, is not very variable, except for the roll of the base-pairs. The hexamer molecular dynamics trajectories have been used to test the restrained parameter crystallographic refinement model for nucleic acids. X-ray diffraction intensities corresponding to observed diffraction data were computed. The average structures obtained from the simulations were then refined against the calculated intensities, using a restrained least-squares program developed for nucleic acids in order to analyse the effects of the refinement model on the derived quantities. In general, the temperature dependence of the atomic fluctuations determined directly from the refined Debye-Waller factors is in reasonably good agreement with the results obtained by calculating the atomic fluctuations directly from the Z-DNA molecular dynamics trajectories. The agreement is best for refinement of temperature factors without restraints. At the highest temperature studied (300 K), the effect of the refinement on the most mobile atoms (phosphates) is to significantly reduce the mean-square atomic fluctuations estimated from the refined Debye-Waller factors below the actual values (less than (delta r)2 greater than congruent to 0.5 A2). Analysis of the temperature-dependence of the mean-square atomic fluctuations provides information concerning the conformational potential within which the atoms move. The calculated temperature-dependence and anharmonicity of the Z-DNA helix are compared with the results observed for proteins. The average structures from the simulations were refined against the experimental X-ray intensities. It is found that low-temperature molecular dynamics simulations provide a useful tool for optimizing the refinement of X-ray structures.     

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.     

Microsecond scale replica exchange molecular dynamic simulation of villin headpiece: an insight into the folding landscape

Reaching the experimental time scale of millisecond is a grand challenge for protein folding simulations. The development of advanced Molecular Dynamics techniques like Replica Exchange Molecular Dynamics (REMD) makes it possible to reach these experimental timescales. In this study, an attempt has been made to reach the multi microsecond simulation time scale by carrying out folding simulations on a three helix bundle protein, Villin, by combining REMD and Amber United Atom model. Twenty replicas having different temperatures ranging from 295 K to 390 K were simulated for 1.5 μs each. The lowest Root Mean Square Deviation (RMSD) structure of 2.5 ? was obtained with respect to native structure (PDB code 1VII), with all the helices formed. The folding population landscapes were built using segment-wise RMSD and Principal Components as reaction coordinates. These analyses suggest the two-stage folding for Villin. The combination of REMD and Amber United Atom model may be useful to understand the folding mechanism of various fast folding proteins.     

Characterization of molecular structures and properties of polyurethanes using molecular dynamics simulations

Thermotropic polyurethanes with mesogenic groups in side chains were prepared from two diisocyanates and four diols with stoichiometric ratios of reactive isocyanate (NCO) and hydroxy (OH) groups. Their thermal behavior was determined by differential scanning calorimetry. The effect of structure modifications of the diisocyanates and diols, in particular changes in the mesogen, were investigated. Introduction of mesogenic segments into the polymers suppresses the ordering. Stiff end substituents (phenyl and alkoxy groups) of the mesogens stabilize the mesophases to such an extent that the negative influence of long polymer chains is compensated and the liquid-crystalline properties are recovered. All-atom molecular dynamics simulations in the Cerius² modeling environment were carried out to characterize the structures of the polymers. Analysis of the dynamic trajectories at 20, 100, 120 and 170 °C revealed changes in conformation of macromolecules, which correlate with DSC measurements.Figure Example of structure relaxation of D4/TDI molecule at indicated simulation times (temperature 20 °C): a complete structure; b backbone structure; c top view of molecule