Exploration of the secondary structure specific differential solvation dynamics between the native and molten globule states of the protein HP-36

Recent experiments have shown that the time dependence of fluorescence Stokes shift of a chromophore is substantially different when the chromophore is located in a molten globule (MG) state and in the native state of the same protein. To understand the origin of this difference, particularly the role of water in the differential solvation of the protein in the native and the MG states, we have carried out fully atomistic molecular dynamics simulations with explicit water of a partially unfolded MG state of the protein HP-36 and compared the results with the solvation dynamics of the protein in the folded native state. It is observed that the polar solvation dynamics of the three helical segments of the protein is influenced in a nonuniform heterogeneous manner in the MG state. While the equilibrium solvation time correlation function for helix-3 has been found to relax faster in the MG state as compared to that in the native state, the decay of the corresponding function for the other two helices slows down in the MG state. A careful analysis shows that the origin of such heterogeneous relative solvation behavior lies in the differential location of the polar probe residues and their exposure to bulk solvent. We find a significant negative cross-correlation between the contribution (to the solvation energy of a tagged amino acid residue) of water and the other groups of the protein, indicating a competing role in solvation. The sensitivity of solvation dynamics to the secondary structure and the immediate environment can be used to discriminate the partially unfolded and folded states. These results therefore should be useful in explaining recent solvation dynamics experiments on native and MG states of proteins.     

Sensitivity of polar solvation dynamics to the secondary structures of aqueous proteins and the role of surface exposure of the probe

The structure and dynamics of water around a protein is expected to be sensitive to the details of the adjacent secondary structure of the protein. In this article, we explore this sensitivity by calculating both the orientational dynamics of the surface water molecules and the equilibrium solvation time correlation function of the polar amino acid residues in each of the three helical segments of the protein HP-36, using atomistic molecular dynamics simulations. The solvation dynamics of polar amino acid residues in helix-2 is found to be faster than that of the other two helices (the average time constant is smaller by a factor of 2), although the interfacial water molecules around helix-2 exhibit much slower orientational dynamics than that around the other two helices. A careful analysis shows that the origin of such a counterintuitive behavior lies in the dependence of the solvation time correlation function on the surface exposure of the probe-the more exposed is the probe, the faster the solvation dynamics. We discuss that these results are useful in explaining recent solvation dynamics experiments.     

Dynamics of water in the hydration layer of a partially unfolded structure of the protein HP-36

Atomistic molecular dynamics simulations of the folded native structure and a partially unfolded molten globule structure of the protein villin headpiece subdomain or HP-36 have been carried out with explicit solvent to explore the effects of unfolding on the dynamical behavior of water present in the hydration layers of different segments (three alpha-helices) of the protein. The calculations revealed that the unfolding of helix-2 influences the translational and rotational motions of water present in the hydration layers of the three helices in a heterogeneous manner. It is observed that a correlation exists between the unfolding of helix-2 and the microscopic kinetics of protein-water hydrogen bonds formed by its residues. This in turn has an influence on the rigidity of the hydration layers of the helices in the unfolded structure versus that in the folded native structure. These results should provide a microscopic explanation to recent solvation dynamics experiments on folded native and unfolded structures of proteins.     

Simulation studies of the protein-water interface. II. Properties at the mesoscopic resolution

We report molecular dynamics (MD) simulations of three protein-water systems (ubiquitin, apo-calbindin D(9K), and the C-terminal SH2 domain of phospholipase C-gamma1), from which we compute the dielectric properties of the solutions. Since two of the proteins studied have a net charge, we develop the necessary theory to account for the presence of charged species in a form suitable for computer simulations. In order to ensure convergence of the time correlation functions needed for the analysis, the minimum length of the MD simulations was 20 ns. The system sizes (box length, number of waters) were chosen so that the resulting protein concentrations are comparable to experimental conditions. A dielectric component analysis was carried out to analyze the contributions from protein and water to the frequency-dependent dielectric susceptibility chi(omega) of the solutions. Additionally, an even finer decomposition into protein, two solvation shells, and the remaining water (bulk water) was carried out. The results of these dielectric decompositions were used to study protein solvation at mesoscopic resolution, i.e., in terms of protein, first and second solvation layers, and bulk water. This study, therefore, complements the structural and dynamical analyses at molecular resolution that are presented in the companion paper. The dielectric component contributions from the second shell and bulk water are very similar in all three systems. We find that the proteins influence the dielectric properties of water even beyond the second solvation shell, in agreement with what was observed for the mean residence times of water molecules in protein solutions. By contrast, the protein contributions, as well as the contributions of the first solvation shell, are system specific. Most importantly, the protein and the first water shell around ubiquitin and apo-calbindin are anticorrelated, whereas the first water shell around the SH2 domain is positively correlated.     

Dynamical changes of hemoglobin and its surrounding water during thermal denaturation as studied by quasielastic neutron scattering and temperature modulated differential scanning calorimetry

We have investigated the dynamical behavior of both the protein hemoglobin and its surrounding water during the denaturation process using modulated temperature differential scanning calorimetry, quasielastic neutron scattering, and frequency dependent conductivity measurements. To distinguish between the scattering from the protein and its surrounding water, neutron scattering measurements were performed on both a fully hydrogenated sample as well as a sample where the water and the exchangeable hydrogen atoms on the protein surface were deuterated. The experimental data show that the unfolding and aggregation processes are substantially overlapping in temperature. The unfolding process occurs in the approximate temperature range of 315-345 K, whereas the aggregation process starts around 330-335 K and is completed at 360 K. Furthermore, the results suggest that the secondary structure of the protein unfolds at about 325 K, and that this leads to an increased number of water molecule hydrogen bonded to the protein. Thus, the unfolding of the secondary structure reduces the number of mobile (on the experimental time scale of about 50-100 ps) water molecules. In contrast, the aggregation of protein molecules seems to have a minor effect on the dynamics of its surrounding water. In the case of the protein dynamics there are competing effects from unfolding and aggregation. The unfolding process increases the flexibility of the protein, whereas the initial aggregation reduces its dynamics. The conductivity seems to be negatively affected by both reduced water mobility and an aggregation of protein molecules.     

Solvation dynamics of a protein in the pre molten globule state

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.     

Polar solvation dynamics of lysozyme from molecular dynamics studies

The solvation dynamics of a protein are believed to be sensitive to its secondary structures. We have explored such sensitivity in this article by performing room temperature molecular dynamics simulation of an aqueous solution of lysozyme. Nonuniform long-time relaxation patterns of the solvation time correlation function for different segments of the protein have been observed. It is found that relatively slower long-time solvation components of the α-helices and β-sheets of the protein are correlated with lower exposure of their polar probe residues to bulk solvent and hence stronger interactions with the dynamically restricted surface water molecules. These findings can be verified by appropriate experimental studies.     

Structural Analysis of a Helical Peptide Unfolding Pathway

The analysis of the folding mechanism in peptides adopting well‐defined secondary structure is fundamental to understand protein folding. Herein, we describe the thermal unfolding of a 15‐mer vascular endothelial growth factor mimicking α‐helical peptide (QK_L10A) through the combination of spectroscopic and computational analyses. In particular, on the basis of the temperature dependencies of QK_L10A H_α chemical shifts we show that the first phase of the thermal helix unfolding, ending at around 320 K, involves mainly the terminal regions. A second phase of the transition, ending at around 333 K, comprises the central helical region of the peptide. The determination of high‐resolution QK_L10A conformational preferences in water at 313 K allowed us to identify, at atomic resolution, one intermediate of the folding–unfolding pathway. Molecular dynamics simulations corroborate experimental observations detecting a stable central helical turn, which represents the most probable site for the helix nucleation in the folding direction. The data presented herein allows us to draw a folding–unfolding picture for the small peptide QK_L10A compatible with the nucleation–propagation model. This study, besides contributing to the basic field of peptide helix folding, is useful to gain an insight into the design of stable helical peptides, which could find applications as molecular scaffolds to target protein–protein interactions.     

A molecular dynamics study of the correlations between solvent-accessible surface, molecular volume, and folding state

We analyzed the correlations between molecular volume, solvent-accessible surface, and folding state (secondary structure content) for unfolded conformers of alpha (holo- and apomyoglobin) and beta (retinal-binding protein) proteins and a small water-soluble alanine-rich alpha-helical peptide. Conformers with different degrees of folding were obtained using molecular dynamics at constant temperature and pressure with implicit solvent (dielectric constant adjustment) for all four systems and with explicit solvent for the single helix peptide. Our results support the view that unfolded conformations are not necessary extended, that volume variation is not a good indication of folding state and that the simple model of water penetrating the interior of the protein does not explain the increase in volume upon unfolding.     

Pressure perturbation calorimetric studies of the solvation properties and the thermal unfolding of proteins in solution--experiments and theoretical interpretation

We used pressure perturbation calorimetry (PPC), a relatively new and efficient technique, to study the solvation and volumetric properties of amino acids and peptides as well as of proteins in their native and unfolded state. In PPC, the coefficient of thermal expansion of the partial volume of the protein is deduced from the heat consumed or produced after small isothermal pressure jumps, which strongly depends on the interaction of the protein with the solvent or cosolvent at the protein-solvent interface. Furthermore, the effects of various chaotropic and kosmotropic cosolvents on the volume and expansivity changes of proteins were measured over a wide concentration range with high precision. Depending on the type of cosolvent and its concentration, specific differences were found for the solvation properties and unfolding behaviour of the proteins, and the volume change upon unfolding may even change sign. To yield a molecular interpretation of the different terms contributing to the partial protein volume and its temperature dependence, and hence a better understanding of the PPC data, molecular dynamics computer simulations on SNase were also carried out and compared with the experimental data. The PPC studies introduced aim to obtain more insight into the basic thermodynamic properties of protein solvation and volume effects accompanying structural transformations of proteins in various cosolvents on one hand, as these form the basis for understanding their physiological functions and their use in drug designing and formulations, but also to initiate further valuable applications in studies of other biomolecular and chemical systems.     

Computer simulation studies of Newton black films

We study via molecular dynamics simulations thin films (Newton black films, NBF) consisting of water coated with sodium dodecyl sulfate (SDS) surfactants. We analyze in detail the film properties (distribution of particles, pair correlation functions, roughness of the film, tilt angle of the hydrocarbon chain, electron density profiles, and mobility of water molecules) as a function of water content in the film core (i.e., film thickness, H). Our simulations indicate that water is part of the bilayer structure as solvation water. We estimate that around 2.25 water molecules per surfactant are part of this solvation structure. The structural analysis of the NBF shows that the headgroups exhibit a high degree of in-plane ordering. We find evidence for the existence of cavities in the monolayer, where only water is present. The basic structure of the monolayer is conserved down to water contents of the order of 4 water molecules per surfactant (H approximately equal to 11 A). The computed monolayer roughness for the present model is 2.5 A, in good agreement with the experimental data. We find that the roughness is very sensitive to the details of the interatomic potentials. Water mobility calculations emphasize the sluggish dynamics of very thin NBF. Diffusion coefficients of water in the lateral direction strongly decrease with film thickness. We find that the typical mean squared displacement of water in the direction normal to the bilayer is between 9 and 80 A2. Overall, our results indicate that the equilibrium SDS Newton black films studied in the X-ray experiments contain from 2 to 4 water molecules per surfactant.     

Understanding the molecular mechanism of enzyme dynamics of ribonuclease A through protonation/deprotonation of HIS48

Molecular dynamics simulation is carried out to investigate the enzyme dynamics of RNase A with the HIS48 in three different states (HIP48 (protonated), HID48 (deprotonated), and H48A mutant). Insights derived from the current theoretical study, combined with the available experimental observations, enabled us to provide a microscopic picture for the efficient enzyme dynamics. Specifically, in the "closed" state or HIP48, the N-terminal hinge loop is intact and the enzyme remains in a relatively stable conformation which is preferred for catalytic reaction. Deprotonation of HIS48 induces the denaturing of this hinge-loop into a 3(10)-helix, causing it to break the original interaction network around the loop-1 and drive the partial unfolding of the N-terminal. The enhanced dynamic motion of the N-terminal helix facilitates the release of the catalytic product (the rate limiting step) and speeds up the overall catalytic process. The current study established that HIS49 acts as a modulator for the transformation of conformational states through the perturbing of hydrogen bond networks across loop-1, the N-terminal helix, and other residues nearby. Our study suggests that HIS48 may also serve to transport loop-1's kinetic energy to the reaction center.     

Ultrafast solvation dynamics of human serum albumin: correlations with conformational transitions and site-selected recognition

Human serum albumin, the most abundant protein found in blood plasma, transports a great variety of ligands in the circulatory system and undergoes reversible conformational transitions over a wide range of pH values. We report here our systematic studies of solvation dynamics and local rigidity in these conformations using a single intrinsic tryptophan (W214) residue as a local molecular probe. With femtosecond resolution, we observed a robust bimodal distribution of time scales for all conformational isomers. The initial solvation occurs in several picoseconds, representing the local librational/rotational motions, followed by the dynamics, in the tens to hundreds of picoseconds, which result from the more bonded water in the tryptophan crevice. Under the physiological condition of neutral pH, we measured approximately 100 ps for the decay of the solvation correlation function and observed a large wobbling motion at the binding site that is deeply buried in a crevice, revealing the softness of the binding pocket and the large plasticity of the native structure. At acidic pH, the albumin molecule transforms to an extended conformation with a large charge distribution at the surface, and a similar temporal behavior was observed. However, at the basic pH, the protein opens the crevice and tightens its globular structure, and we observed significantly faster dynamics, 25-45 ps. These changes in the solvation dynamics are correlated with the conformational transitions and related to their structural integrity.     

Structural information content and Lyapunov exponent calculation in protein unfolding

A lattice model with Monte Carlo dynamics is used to carry out computer simulations of protein dynamics on a four α-helix bundle. The interaction energies in the model can be set so that either the helix bundle structure remains relatively stable or changed so that it unfolds. The computer model produces output that simulates experimental measurements relating to the structure. We show how this output can be used with analytical techniques of nonlinear dynamics to obtain important information about the complex underlying protein dynamics. Time-delay reconstruction plots of structural parameters of unfolding bundles resemble strange attractors in a space of dimension 3–4. We calculate Lyapunov exponents for these unfolding runs and find positive Lyapunov exponents implying chaotic dynamics. For stable runs the Lyapunov exponents are close to zero. We use these Lyapunov exponents to calculate the rate of loss of structural information during the unfolding process and show how the approach may be useful for investigating the folding dynamics of proteins. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 2761–2769, 1998     

FACTS: Fast analytical continuum treatment of solvation

An efficient method for calculating the free energy of solvation of a (macro)molecule embedded in a continuum solvent is presented. It is based on the fully analytical evaluation of the volume and spatial symmetry of the solvent that is displaced from around a solute atom by its neighboring atoms. The two measures of solvent displacement are combined in empirical equations to approximate the atomic (or self) electrostatic solvation energy and the solvent accessible surface area. The former directly yields the effective Born radius, which is used in the generalized Born (GB) formula to calculate the solvent-screened electrostatic interaction energy. A comparison with finite-difference Poisson data shows that atomic solvation energies, pair interaction energies, and their sums are evaluated with a precision comparable to the most accurate GB implementations. Furthermore, solvation energies of a large set of protein conformations have an error of only 1.5%. The solvent accessible surface area is used to approximate the nonpolar contribution to solvation. The empirical approach, called FACTS (Fast Analytical Continuum Treatment of Solvation), is only four times slower than using the vacuum energy in molecular dynamics simulations of proteins. Notably, the folded state of structured peptides and proteins is stable at room temperature in 100-ns molecular dynamics simulations using FACTS and the CHARMM force field.     

Reorientational dynamics of p-sulfonatocalix[4]arene and of its La(III) complex in water

The reorientational dynamics of p-sulfonatocalix[4]arene and of its La(III) complex in deuterated water were studied by 1H NMR longitudinal relaxation rates. It is shown that the relaxation is purely dipolar in the non-extreme narrowing regime. The distance between the geminal protons could be determined from the NMR data, giving good agreement with the values generally used in correlation time calculations. The correlation times show an Arrhenius behaviour in good agreement with previously reported data from 13C measurements for a similar uncomplexed calixarene. The Arrhenius energies of activation are identical for the uncomplexed and the complexed calixarenes, suggesting a reorientational motion strongly dependent on the structure of the water cage around the complex. This is also in agreement with a complexation of the La(III) cation in the second sphere of solvation of the sulfonate groups, as shown by molecular dynamics simulations.     

Dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations

Atomistic molecular dynamics simulations are used to investigate the mechanism by which the antifreeze protein from the spruce budworm, Choristoneura fumiferana, binds to ice. Comparison of structural and dynamic properties of the water around the three faces of the triangular prism-shaped protein in aqueous solution reveals that at low temperature the water structure is ordered and the dynamics slowed down around the ice-binding face of the protein, with a disordering effect observed around the other two faces. These results suggest a dual role for the solvation water around the protein. The preconfigured solvation shell around the ice-binding face is involved in the initial recognition and binding of the antifreeze protein to ice by lowering the barrier for binding and consolidation of the protein:ice interaction surface. Thus, the antifreeze protein can bind to the molecularly rough ice surface by becoming actively involved in the formation of its own binding site. Also, the disruption of water structure around the rest of the protein helps prevent the adsorbed protein becoming covered by further ice growth.     

NMR studies of the dynamics of a bifunctional rhodamine probe attached to troponin C

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.     

Choline Chloride as a Nano-Crowder Protects HP-36 from Urea-Induced Denaturation: Insights from Solvent Dynamics and Protein-Solvent Interactions

Urea at sufficiently high concentration unfolds the secondary structure of proteins leading to denaturation. In contrast, choline chloride (ChCl) and urea, in 1 : 2 molar ratio, form a deep eutectic mixture, a liquid at room temperature, protecting proteins from denaturation. In order to get a microscopic picture of this phenomenon, we perform extensive all-atom molecular dynamics simulations on a model protein, HP-36. Based on our calculation of Kirkwood-Buff integrals, we analyze the relative accumulation of urea and ChCl around the protein. Additional insights are drawn from the translational and rotational dynamics of solvent molecules and hydrogen bond auto-correlation functions. In the presence of urea, water shows slow subdiffusive dynamics around the protein owing to a strong interaction of water with the backbone atoms. Urea also shows subdiffusive motion. The addition of ChCl further slows down the dynamics of urea, restricting its accumulation around the protein backbone. Adding to this, choline cations in the first solvation shell of the protein show the strongest subdiffusive behavior. In other words, ChCl acts as a nano-crowder by excluding urea from the protein backbone and thereby slowing down the dynamics of water around the protein. This prevents the protein from denaturation and makes it structurally rigid, which is supported by the smaller radius of gyration and root mean square deviation values of HP-36.