Chemical denaturants inhibit the onset of dewetting

The mechanism by which the aqueous cosolvents guanidinium chloride and urea denature proteins is a matter of controversy. Here, we use all-atom molecular dynamics simulations to study the effect of both denaturants on the dewetting of water confined between nanoseparated hydrophobic plates. It is found that the denaturants inhibit the onset of dewetting, so that it occurs at shorter interplate distances than in pure water. Our results support a role for urea and guanidinium in assisting in the solvation of nonpolar surfaces, thereby weakening hydrophobic effects known to be important for protein stability.     

The hydrophobic effect: molecular dynamics simulations of water confined between extended hydrophobic and hydrophilic surfaces

Structural and dynamic properties of water confined between two parallel, extended, either hydrophobic or hydrophilic crystalline surfaces of n-alkane C(36)H(74) or n-alcohol C(35)H(71)OH, are studied by molecular dynamics simulations. Electron density profiles, directly compared with corresponding experimental data from x-ray reflectivity measurements, reveal a uniform weak de-wetting characteristic for the extended hydrophobic surface, while the hydrophilic surface is weakly wetted. These microscopic data are consistent with macroscopic contact angle measurements. Specific water orientation is present at both surfaces. The ordering is characteristically different between the surfaces and of longer range at the hydrophilic surface. Furthermore, the dynamic properties of water are different at the two surfaces and different from the bulk behavior. In particular, at the hydrophobic surface, time-correlation functions reveal that water molecules have characteristic diffusive behavior and orientational ordering due to the lack of hydrogen bonding interactions with the surface. These observations suggest that the altered dynamical properties of water in contact with extended hydrophobic surfaces together with a partial drying of the surfaces are more indicative of the hydrophobic effect than structural ordering, which we suggest to be independent of surface topology.     

The native ensemble and folding of a protein molten-globule: functional consequence of downhill folding

The continually emerging functional significance of intrinsic disorder and conformational flexibility in proteins has challenged the long-standing dogma of a well-defined structure contributing to a specific function. Molten-globular states, a class of proteins with significant secondary-structure but a fluid hydrophobic core, is one such example. They have however been difficult to characterize due to the complexity of experimental data and lack of computational avenues. Here, we dissect the folding mechanism of the α-helical molten-globular protein NCBD from three fundamentally different approaches: statistical-mechanical variable barrier model, C(α)-based Gō-model and explicit water all-atom molecular dynamics (MD) simulations. We find that NCBD displays the characteristics of a one-state globally downhill folder but is significantly destabilized. Using simulation techniques, we generate a highly constrained but a heterogeneous native ensemble of the molten-globule for the first time that is consistent with experimental data including small angle X-ray scattering (SAXS), circular dichroism (CD), and nuclear magnetic resonance (NMR). The resulting native ensemble populates conformations reported in other bound-forms providing direct evidence to the mechanism of conformational selection for binding multiple partners in this domain. Importantly, our simulations reveal a connection between downhill folding and large conformational flexibility in this domain that has been evolutionarily selected and functionally exploited resulting in large binding promiscuity. Finally, the multimodel approach we employ here serves as a powerful methodology to study mechanisms and suggests that the thermodynamic features of molten-globules fall within the array of folding mechanisms available to small single-domain proteins.     

Molecular dynamics study of the properties of capsaicin in an 1-octanol/water system

Molecular dynamics simulations were performed to study the behavior of capsaicin in an 1-octanol/water system at 298 K and 1 bar. Capsaicin is the pungent chemical found in chili pepper that stimulates our sensory system resulting in a burning, pain sensation. In the first step toward investigating the activity of capsaicin, we have used two molecular representations for capsaicin based on the OPLS force field: all-atom and united-atom models. The octanol/water mixture was selected as a model system to determine the hydrophobic and hydrophilic properties of capsaicin by analyzing equilibrium, structural, and dynamic properties from the simulations. Our simulations showed that capsaicin preferentially partitions to the octanol phase, with its hydrocarbon segment oriented with that in octanol, while the polar part remains exposed to the aqueous phase. The simulations with the all-atom and united-atom models yielded similar results.     

A role for confined water in chaperonin function

Chaperonins engulf other proteins and accelerate their folding by an unknown mechanism. Here, we combine all-atom molecular dynamics simulations with data from experimental assays of the activity of the bacterial chaperonin GroEL to demonstrate that a chaperonin's ability to facilitate folding is correlated with the affinity of its interior surface for water. Our results suggest a novel view of the behavior of confined water for models of in vivo protein folding scenarios.     

The equilibrium properties and folding kinetics of an all-atom Go model of the Trp-cage

The ultrafast-folding 20-residue Trp-cage protein is quickly becoming a new benchmark for molecular dynamics studies. Already several all-atom simulations have probed its equilibrium and kinetic properties. In this work an all-atom Go model is used to accurately represent the side-chain packing and native atomic contacts of the Trp-cage. The model reproduces the hallmark thermodynamics cooperativity of small proteins. Folding simulations observe that in the fast-folding dominant pathway, partial alpha-helical structure forms before hydrophobic core collapse. In the slow-folding secondary pathway, partial core collapse occurs before helical structure. The slow-folding rate of the secondary pathway is attributed to the loss of side-chain rotational freedom, due to the early core collapse, which impedes the helix formation. A major finding is the observation of a low-temperature kinetic intermediate stabilized by a salt bridge between residues Asp-9 and Arg-16. Similar observations [R. Zhou, Proc. Natl. Acad. Sci. U.S.A. 100, 13280 (2003)] were reported in a recent study using an all-atom model of the Trp-cage in explicit water, in which the salt-bridge stabilized intermediate was hypothesized to be the origin of the ultrafast-folding mechanism. A theoretical mutation that eliminates the Asp-9-Arg-16 salt bridge, but leaves the residues intact, is performed. Folding simulations of the mutant Trp-cage observe a two-state free-energy landscape with no kinetic intermediate and a significant decrease in the folding rate, in support of the hypothesis.     

Molecular dynamics with the united-residue model of polypeptide chains. II. Langevin and Berendsen-bath dynamics and tests on model alpha-helical systems

The implementation of molecular dynamics (MD) with our physics-based protein united-residue (UNRES) force field, described in the accompanying paper, was extended to Langevin dynamics. The equations of motion are integrated by using a simplified stochastic velocity Verlet algorithm. To compare the results to those with all-atom simulations with implicit solvent in which no explicit stochastic and friction forces are present, we alternatively introduced the Berendsen thermostat. Test simulations on the Ala(10) polypeptide demonstrated that the average kinetic energy is stable with about a 5 fs time step. To determine the correspondence between the UNRES time step and the time step of all-atom molecular dynamics, all-atom simulations with the AMBER 99 force field and explicit solvent and also with implicit solvent taken into account within the framework of the generalized Born/surface area (GBSA) model were carried out on the unblocked Ala(10) polypeptide. We found that the UNRES time scale is 4 times longer than that of all-atom MD simulations because the degrees of freedom corresponding to the fastest motions in UNRES are averaged out. When the reduction of the computational cost for evaluation of the UNRES energy function is also taken into account, UNRES (with hydration included implicitly in the side chain-side chain interaction potential) offers about at least a 4000-fold speed up of computations relative to all-atom simulations with explicit solvent and at least a 65-fold speed up relative to all-atom simulations with implicit solvent. To carry out an initial full-blown test of the UNRES/MD approach, we ran Berendsen-bath and Langevin dynamics simulations of the 46-residue B-domain of staphylococcal protein A. We were able to determine the folding temperature at which all trajectories converged to nativelike structures with both approaches. For comparison, we carried out ab initio folding simulations of this protein at the AMBER 99/GBSA level. The average CPU time for folding protein A by UNRES molecular dynamics was 30 min with a single Alpha processor, compared to about 152 h for all-atom simulations with implicit solvent. It can be concluded that the UNRES/MD approach will enable us to carry out microsecond and, possibly, millisecond simulations of protein folding and, consequently, of the folding process of proteins in real time.     

Ab initio folding of helix bundle proteins using molecular dynamics simulations

We have demonstrated that ab initio fast folding simulations at 400 K using a GB implicit solvent model with an all-atom based force field can describe the spontaneous formation of nativelike structures for the 36-residue villin headpiece and the 46-residue fragment B of Staphylococcal protein A. An implicit solvent model combined with high-temperature MD makes it possible to perform direct folding simulations of small- to medium-sized proteins by reducing the computational requirements tremendously. In the early stage of folding of the villin headpiece and protein A, initial hydrophobic collapse and rapid formation of helices were found to play important roles. For protein A, the third helix forms first in the early stage of folding and exhibits higher stability. The free energy profiles calculated from the folding simulations suggested that both of the helix-bundle proteins show a two-state thermodynamic behavior and protein A exhibits rather broad native basins.     

A generic rotamer model to explain the temperature dependence of BSA protein fluorescence

Protein fluorescence signals essential information about the conformational dynamics of proteins. Different types of intrinsic fluorophores reflect different protein local or global structural changes. Bovine Serum Albumin (BSA) is a transport protein that contains two intrinsic fluorophores: Tryptophan134 (Trp134) and Tryptophan213 (Trp213). This protein displays an interesting temperature dependence of the tryptophan fluorescence. However, the molecular mechanism of the temperature dependence is still unclear. In this work, we propose a generic rotamer model to explain this phenomenon. The model assumes the presence of rotamer-specific fluorescence lifetimes. The fluorescence temperature dependence is caused by the population shifts between different rotamers due to thermal effects. As a proof of concept, we show that the tryptophan's two fluorescence lifetimes (𝜏₁ = 0.4–0.5 ns and 𝜏₂ = 2-4 ns) are sufficient to qualitatively explain the fluorescence intensity change at different temperatures, both in buffer solution (water) and in the protein. To computationally verify our rotamer hypothesis, we use an all-atom molecular dynamics simulation to study the effects of temperature on the two tryptophans' rotamer dynamics. The simulations show that Trp134 is more sensitive to temperature, consistent with experimental observations. Overall, the results support that the temperature dependence of fluorescence in the protein BSA is due to local conformational changes at the residue level. This work sheds light on the relationship between tryptophan's rotamer dynamics and its ability to fluorescence.     

Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.     

Refinement of the primary hydration shell model for molecular dynamics simulations of large proteins

A realistic representation of water molecules is important in molecular dynamics simulation of proteins. However, the standard method of solvating biomolecules, that is, immersing them in a box of water with periodic boundary conditions, is computationally expensive. The primary hydration shell (PHS) method, developed more than a decade ago and implemented in CHARMM, uses only a thin shell of water around the system of interest, and so greatly reduces the computational cost of simulations. Applying the PHS method, especially to larger proteins, revealed that further optimization and a partial reworking was required and here we present several improvements to its performance. The model is applied to systems with different sizes, and both water and protein behaviors are compared with those observed in standard simulations with periodic boundary conditions and, in some cases, with experimental data. The advantages of the modified PHS method over its original implementation are clearly apparent when it is applied to simulating the 82 kDa protein Malate Synthase G. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

海藻糖和氨基酸之间相互作用的分子动力学模拟

虽然海藻糖已经广泛用于蛋白质稳定性研究,但海藻糖稳定蛋白质的作用机理尚不清晰. 本文利用全原子分子动力学模拟研究了20种常见氨基酸和海藻糖之间的分子机理. 结果表明,所有氨基酸,尤其是极性和带电氨基酸,均优先与水分子结合. 相反,仅有疏水性氨基酸与海藻糖发生相互作用,尤其是芳香族和疏水性氨基酸的侧链更易于和海藻糖接触. 所有氨基酸的主链与水分子接触的趋势一致. 虽然氨基酸和海藻糖与水之间均形成氢键,但氨基酸和海藻糖之间的氢键相互作用要弱于氨基酸和水之间的氢键相互作用. 上述分子模拟的结果对于海藻糖稳定蛋白质作用机理的解析及高效蛋白质稳定剂的理性设计具有非常重要的理论指导意义.     

海藻糖和氨基酸之间相互作用的分子动力学模拟

虽然海藻糖已经广泛用于蛋白质稳定性研究,但海藻糖稳定蛋白质的作用机理尚不清晰.本文利用全原子分子动力学模拟研究了20种常见氨基酸和海藻糖之间的分子机理.结果表明,所有氨基酸,尤其是极性和带电氨基酸,均优先与水分子结合.相反,仅有疏水性氨基酸与海藻糖发生相互作用,尤其是芳香族和疏水性氨基酸的侧链更易于和海藻糖接触.所有氨基酸的主链与水分子接触的趋势一致.虽然氨基酸和海藻糖与水之间均形成氢键,但氨基酸和海藻糖之间的氢键相互作用要弱于氨基酸和水之间的氢键相互作用.上述分子模拟的结果对于海藻糖稳定蛋白质作用机理的解析及高效蛋白质稳定剂的理性设计具有非常重要的理论指导意义.     

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.     

蛋白折叠中的暂态结构与表面水分子慢尺度动力学

蛋白表面水的慢尺度动力学行为往往被认为与蛋白的结构稳定性、功能以及折叠过程有关, 但在分子水平上, 还不清楚水分子的慢尺度动力学如何参与蛋白折叠过程. 以Trp-cage蛋白作为个案, 本文利用40条100 ns(总长4 μs)的全原子分子动力学轨迹,分析了蛋白折叠过程中蛋白表面水分子的停留行为,并探究影响蛋白表面水分子慢尺度行为的微观因素. 结果发现, 即使在蛋白折叠过程中蛋白拓扑结构变化很大, 残基之间也会形成稳定的局部暂态结构. 这些结构为水分子提供饱和、稳定的氢键, 通过与水分子之间的极性相互作用, 以及凹形的几何结构, 约束水分子长时停留, 我们称之为“停留中心”. 停留中心的形成是引起水分子慢尺度行为的重要因素. 另外, 停留中心的分布与蛋白折叠的进程有密切关系, 特别地, 在折叠轨迹中, 疏水核周围的残基组成了一个主要的停留中心. 研究结果不但有助于解释水分子慢尺度特征行为的来源, 还可以为实验中通过研究水分子在蛋白附近的慢尺度行为, 揭示蛋白折叠过程中的关键步骤提供一些启发.     

Contribution of omega-3 fatty acids to the thermodynamics of membrane protein solvation

Recent NMR experiments and molecular dynamics simulations have indicated that rhodopsin is preferentially solvated by omega-3 fatty acids compared to saturated chains. However, to date no physical theory has been advanced to explain this phenomenon. The present work presents a novel thermodynamic explanation for this preferential solvation based on statistical analysis of 26,100 ns all-atom molecular dynamics simulations of rhodopsin in membranes rich in polyunsaturated chains. The results indicate that the preferential solvation by omega-3 chains is entropically driven; all chains experience an entropic penalty when associating with the protein, but the penalty is significantly larger for saturated chains.     

Atomic-level investigations on the amyloid-β dimerization process and its driving forces in water

We report the spontaneous dimerization process of the full-length Aβ42 proteins in water by using unguided, fully atomistic, explicit-water molecular dynamics simulations. Based on the thermodynamic analysis, we demonstrate that Aβ42 dimerization in water occurs via a two-step nucleation-accommodation mechanism driven by water-induced force and by protein internal force, respectively.     

Atomistic mechanism of protein denaturation by urea

Effects of urea on protein stability have been studied from all-atom molecular dynamics simulations of ubiquitin, G311 protein, and immunoglobulin binding domain (B1) of streptococcal protein G (GB1) in water and 8 M aqueous urea solution. The mechanism of the change in the solvent environment and the early events in protein unfolding by urea have been identified with emphasis on the change in the interactions of hydrophilic and hydrophobic parts of the protein by calculating the potential of mean force (PMF). Urea replaces the protein-protein and protein-water contacts by forming stronger contacts with the protein, which is indicated by the longer survival times of the protein-urea hydrogen bonds.     

Modeling real dynamics in the coarse-grained representation of condensed phase systems

This work presents a systematic multiscale methodology to provide a more faithful representation of real dynamics in coarse-grained molecular simulation models. The theoretical formalism is based on the recently developed multiscale coarse-graining (MS-CG) method [S. Izvekov and G. A. Voth, J. Phys. Chem. B. 109, 2469 (2005); J. Chem. Phys. 123, 134105 (2005)] and relies on the generalized Langevin equation approach and its simpler Langevin equation limit. The friction coefficients are determined in multiscale fashion from the underlying all-atom molecular dynamics simulations using force-velocity and velocity-velocity correlation functions for the coarse-grained sites. The diffusion properties in the resulting CG Brownian dynamics simulations are shown to be quite accurate. The time dependence of the velocity autocorrelation function is also well-reproduced relative to the all-atom model if sufficient resolution of the CG sites is implemented.