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.     

Nature of the cytochrome c molten globule

We have employed fluorescence energy transfer (FET) kinetics to probe unfolded and molten globule states of five dansyl (Dns) variants of Saccharomyces cerevisiae iso-1 cytochrome c. The covalently bound Fe(III) heme group quenches Dns fluorescence by energy transfer; measurements of FET kinetics yielded distributions of D-A distances (P(r)) for these states. The P(r) distributions and corresponding mean force potentials (U(r)) show that the cytochrome c molten globule is a highly structured state with a substantial number of native interactions. Wide P(r) distributions directly reflect the dynamic nature and conformational diversity of this molten globule. P(r) distributions for the "burst-phase" refolding intermediate suggest that the equilibrium cytochrome c molten globule is not a suitable model for early intermediates formed during protein refolding.     

Site-specific hydration dynamics in the nonpolar core of a molten globule by dynamic nuclear polarization of water

Water-protein interactions play a direct role in protein folding. The chain collapse that accompanies protein folding involves extrusion of water from the nonpolar core. For many proteins, including apomyoglobin (apoMb), hydrophobic interactions drive an initial collapse to an intermediate state before folding to the final structure. However, the debate continues as to whether the core of the collapsed intermediate state is hydrated and, if so, what the dynamic nature of this water is. A key challenge is that protein hydration dynamics is significantly heterogeneous, yet suitable experimental techniques for measuring hydration dynamics with site-specificity are lacking. Here, we introduce Overhauser dynamic nuclear polarization at 0.35 T via site-specific nitroxide spin labels as a unique tool to probe internal and surface protein hydration dynamics with site-specific resolution in the molten globular, native, and unfolded protein states. The (1)H NMR signal enhancement of water carries information about the local dynamics of the solvent within ～10 ? of a spin label. EPR is used synergistically to gain insights on local polarity and mobility of the spin-labeled protein. Several buried and solvent-exposed sites of apoMb are examined, each bearing a covalently bound nitroxide spin label. We find that the nonpoloar core of the apoMb molten globule is hydrated with water bearing significant translational dynamics, only 4-6-fold slower than that of bulk water. The hydration dynamics of the native state is heterogeneous, while the acid-unfolded state bears fast-diffusing hydration water. This study provides a high-resolution glimpse at the folding-dependent nature of protein hydration dynamics.     

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.     

Structural features of the cytochrome C molten globule revealed by fluorescence energy transfer kinetics

Nonnative states of proteins are involved in a variety of cellular processes, including translocation of proteins across membranes and formation of amyloid fibrils. Probes that report on the structural heterogeneity of a polypeptide ensemble could resolve ambiguities in the classification of these states. Employing fluorescence energy transfer kinetics, we have shown that added anions shift the equilibrium between the compact and extended polypeptide structures that are present during refolding of Saccaromyces cerevisiae iso-1 cytochrome c. Specifically, at high salt concentrations (>/=700 mM), all of the polypeptides are compact with a mean C-terminal fluorophore-heme separation quite close to that in the native protein (25 A).     

熔融ZnCl2结构的分子动力学模拟研究

熔融ZnCl2作为一种离子性共价性参半的典型熔盐, 其近邻结构在实验测量和分子动力学模拟方面均作过一些研究。本文依据新近EXAFS实验结果, 比较了不同的有效势下模拟得到的径向分布函数,表明KDR势可作为一种实用势。并进一步在KDR势模拟产生的瞬态构型基础上, 使用键序参数方法研究了晶态和熔融态ZnCl2中的近邻结构。结果表明, 和晶态ZnCl2一样, 在熔融ZnCl2中存在稳定的Zn/Cl正四面体结构, 但熔态和晶态Zn/Cl近邻结构热波方差σ不同。计算表明300K晶态σ=5.0℃, 613K熔融态σ=12.2℃。也对熔融ZnCl2的网络状结构和宏观输运性质进行了讨论。     

Temperature dependence of anisotropic protein backbone dynamics

The measurement of (15)N NMR spin relaxation, which reports the (15)N-(1)H vector reorientational dynamics, is a widely used experimental method to assess the motion of the protein backbone. Here, we investigate whether the (15)N-(1)H vector motions are representative of the overall backbone motions, by analyzing the temperature dependence of the (15)N-(1)H and (13)CO-(13)C(alpha) reorientational dynamics for the small proteins binase and ubiquitin. The latter dynamics were measured using NMR cross-correlated relaxation experiments. The data show that, on average, the (15)N-(1)H order parameters decrease only by 2.5% between 5 and 30 degrees C. In contrast, the (13)CO-(13)C(alpha) order parameters decrease by 10% over the same temperature trajectory. This strongly indicates that there are polypeptide-backbone motions activated at room temperature that are not sensed by the (15)N-(1)H vector. Our findings are at variance with the common crank-shaft model for protein backbone dynamics, which predicts the opposite behavior. This study suggests that investigation of the (15)N relaxation alone would lead to underestimation of the dynamics of the protein backbone and the entropy contained therein.     

Fast local backbone dynamics of encapsulated ubiquitin

Backbone dynamics of ubiquitin confined within AOT reverse micelles have been evaluated based on analysis of 15N NMR relaxation data. Results indicate that upon encapsulation the protein experiences a slight overall increase in the value of the order parameter, S2, indicating a restriction in the average amplitude of fast local N-H bond vector motion. The largest increases in S2 upon encapsulation were concentrated in the region of beta-sheet 2 and, additionally, at the transitions of secondary structure motifs and loop regions. In addition, statistical analysis of the residue average ratio of the 15N longitudinal and transverse NMR relaxation time constants indicates that chemical exchange contributions to relaxation are consistent with previous aqueous studies. Earlier studies have demonstrated that native protein structure can be maintained in the encapsulated state. These results presented here establish that the dynamical behavior of encapsulated ubiquitin is likewise nativelike and adds important new observations regarding the enhancement of protein stability under confinement.     

Thermodynamic insights into the binding of ANS with the salt induced molten globule states of cytochrome c

The molten globule (MG) state or the A-state of cytochrome c (cyt c) has been induced by addition of salts sodium perchlorate (NaClO₄), sodium thiocyanate (NaSCN), and sodium sulphate (Na₂SO₄) at pH 2.0. Isothermal titration calorimetry (ITC) has been used for determining the energetics of binding of 8-anilino naphthalene sulphonate (ANS) to the salt induced A-state of cyt c, and the accompanying thermodynamic parameters have been analyzed to elucidate the nature of the interactions between ANS and the A-state of cyt c. Temperature dependent studies of the binding process reveal that the binding is not a two state process and there are more than a single type of interactions involved. Addition of a bulky salt tetraethylammonium bromide (TEAB) increases the stoichiometry of binding significantly, with a reduction in the binding affinity at a higher concentration. The results provide quantitative information on the binding of ANS to the salt induced molten globule states of cyt c. It is further inferred that the binding involves a combination of hydrophobic and electrostatic interactions.     

First principles molecular dynamics of molten NaCl

First principles Hellmann-Feynman molecular dynamics (HFMD) results for molten NaCl at a single state point are reported. The effect of induction forces on the structure and dynamics of the system is studied by comparison of the partial radial distribution functions and the velocity and force autocorrelation functions with those calculated from classical MD based on rigid-ion and shell-model potentials. The first principles results reproduce the main structural features of the molten salt observed experimentally, whereas they are incorrectly described by both rigid-ion and shell-model potentials. Moreover, HFMD Green-Kubo self-diffusion coefficients are in closer agreement with experimental data than those predicted by classical MD. A comprehensive discussion of MD results for molten NaCl based on different ab initio parametrized polarizable interionic potentials is also given.     

Molecular dynamics simulation on influence of temperature effect on electro-coalescence behavior of nano-droplets

In electric dehydration of crude oil, the dewatering efficiency can be improved by raising emulsion temperature properly which reduces the viscosity of crude oil. However, it should be noticed that the emulsion temperature does not only affect the emulsion viscosity but also nano-droplets dynamics behavior which impacts the coalescence efficiency either. Therefore the influence of temperature effect on the electro-coalescence of nano-droplets is studied by a molecular dynamics method. The results show that the temperature presents an active or negative effect, depending on the competitive relation between electrostatic interaction and thermal motion. Two stages are distinguished according to the dominant mechanism. During stage I, governed by the electrostatic interaction, lower temperature promotes the polarization and leads to an acceleration of the droplets coalescence, but higher temperature restrains the coalescence process due to molecules thermal motion breaking the polarization process. During stage II, governed by the thermal motion, lower temperature improves the coalescence because of a diffusion effect, but higher temperature deteriorates electro-coalescence because of a violent molecular thermal motion. Additionally, hydrogen bond and radial distribution functions are obtained by statistics to describe droplets micromorphology, which explains the reason why the droplet forms longer chain structure at the critical electric field.     

Structure and dynamics of alpha-lactalbumin adsorbed at a charged brush interface

We have studied the adsorption of alpha-lactalbumin at a planar poly(acrylic acid) (PAA) brush using neutron reflectometry (NR) and total internal reflection fluorescence (TIRF) spectroscopy. The PAA brush has been prepared by spin-coating silicon or quartz plates with a hydrophobic poly(styrene) film and by transferring the copolymer poly(styrene)-poly(acrylic acid) onto the modified surface. In the case of NR, the poly(styrene) film and the poly(styrene) chain ends of the copolymer were perdeuterated in order to generate a high contrast to the non-deuterated PAA brush. alpha-Lactalbumin was chosen as the model protein because it is a relatively small globular protein with a negative net charge at neutral pH-values, as chosen in the experiments. Thus, it is interacting with the PAA brush on the 'wrong' side of its isoelectric point. In addition, the effects of temperature on the volume fraction profile and the reorientational mobility of the protein within the PAA brush were determined. From the analysis of the NR data, it has been found that despite of its negative net charge, alpha-lactalbumin is penetrating into the PAA brush. Its volume fraction profile parallels that of the PAA brush, indicating an exclusive interaction between the protein and the PAA. No protein accumulation is found at the inner poly(styrene) or the outer solution interface of the PAA brush. When increasing the temperature from 20 to 40 degrees C, less protein is adsorbed, suggesting the presence of enthalpic interaction contributions. From the analysis of the TIRF data, a high degree of reorientational mobility of alpha-lactalbumin within a PAA brush can be inferred. The reorientational correlation time of alpha-lactalbumin labeled with the Alexa Fluor 488 dye was found to increase from 5.5 to 32 ns upon adsorption, which can well be explained by the higher viscosity inside the PAA brush. Overall, the results of this study quantify for the first time the molecular details of the unique interaction of a protein on the 'wrong' side of its isoelectric point with a planar charged brush interface. It is concluded that the high mobility of alpha-lactalbumin within a PAA brush can partially be understood by the presence of repulsive electrostatic interactions. There is no 'freezing' of the protein dynamics, which is a precondition for biological activity.     

Study on the coil–globule transition and dynamics of polymer chain confined in slit

The coil–globule transition and dynamics of a lattice self‐avoiding bond fluctuation polymer chain confined in slit are studied by Monte Carlo simulations. The coil–globule transition temperature of polymer chain is increased at intermediate slit height H (H ∼ R_G0 with R_G0 the radius of gyration of polymer in dilute solution) due to the squeeze of the polymer in the repulsive slit, but it is decreased by surface attraction as the polymer is extended along the surface. We have compared the difference between the rotational relaxation time τ_R for the reorientation of end‐to‐end vector and the relaxation time τ for the polymer diffusing over a distance of the size of polymer. We find that τ_R is clearly distinct from τ as they have different scaling exponents in their slit height‐dependent behaviors and for the polymer in the extended coil state, that is, α_R > α. And both exponents increase with an increase in the intrapolymer attraction and surface attraction. However, these scaling relations are destroyed by strong surface attraction when the polymer is adsorbed on surfaces. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 1053–1062     

Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: spectroscopic and calorimetric approaches

Papain exists in a molten globule (MG) state at pH 2 and in this state protein tends to aggregate in the presence of lower concentrations of guanidine hydrochloride (GuHCl). Such aggregation is prevented if low concentrations of sodium n-alkyl sulfates are also present in the buffer; in addition, stabilization of the protein is also induced. The guanidine hydrochloride and temperature-induced unfolding of papain, in the presence of n-alkyl sulfates, indicate stabilization of the protein as seen from the higher transition midpoints when monitored by fluorescence, circular dichroism, and differential scanning calorimetry. However, a similar phenomenon is not seen under neutral conditions in the presence of n-alkyl sulfate concentrations. The effect of n-alkyl sulfates on the structure of the MG state of papain was utilized to investigate the contribution of hydrophobic interaction to the stability of the MG state. The Td values of the MG states of papain in the presence of n-alkyl sulfates at different concentrations showed substantial variation. The enhancement of Td values at the stability criterion of MG states corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the hydrophobic interactions play important roles in stabilizing and preventing the aggregation of the MG state of papain.     

Molecular dynamics study of the nucleation behavior and lamellar mergence in polyethylene globule crystallization

The site order parameter (SOP) has been adopted to analyze various orders of structure formation and distribution during the crystallization of a polyethylene globule simulated by molecular dynamics. The SOP not only identifies different orders among the structures but also different phases. The results showed that intermediate structures with medium order exist unevenly in the early stage of nucleation, which differs from traditional models. We found that the nucleation relies on crystallinity fluctuations with increasing amplitude, and a baby nucleus with different shape and increasing size suddenly appears in the fluctuations. Once its size is large enough, the baby nucleus becomes the nucleus. In the growth stage, a number of lamellar mergences were observed and their selective behaviors were related to the orientation differences between the lamellae to be merged. The SOP distribution of all atoms in the system during crystallization had two peaks: one for the amorphous phase and the other for the crystalline phase. The intermediate structures with medium orders are located between the two peaks as an order promotion pathway. The data show that the medium order structure fluctuates at the growth front and is not always available, and that the medium order structure existing at the front is not always good for development. It is possibly caused by chain entanglement. Structure fluctuation at the growth front enables the system showing thousands of the most probable configurations to approach the precursor. The growth front is thus active for a while and inactive at other times.     

Probing the structure of lysozyme-carbon-nanotube hybrids with molecular dynamics

Lysozyme has been successfully used to solvate carbon nanotubes (CNT). Extensive molecular dynamics simulations show that 1) a favorite site of adsorption exists, 2) the protein-tube interaction region is located far from the active site, 3) two protein helices act as a tweezer that grips the nanotube, 4) a localized protein re-arrangement hides the tube from the solvent, and 5) aminic and amidic moieties of lysozyme behave similarly to surfactants in the solvation of the tube.     

Communication: a minimal model for the diffusion-relaxation backbone dynamics of proteins

We present a model for the local diffusion-relaxation dynamics of the C(α)-atoms in proteins describing both the diffusive short-time dynamics and the asymptotic long-time relaxation of the position autocorrelation functions. The relaxation rate spectra of the latter are represented by shifted gamma distributions, where the standard gamma distribution describes anomalous slow relaxation in macromolecular systems of infinite size and the shift accounts for a smallest local relaxation rate in macromolecules of finite size. The resulting autocorrelation functions are analytic for any time t ≥ 0. Using results from a molecular dynamics simulation of lysozyme, we demonstrate that the model fits the position autocorrelation functions of the C(α)-atoms exceptionally well and reveals moreover a strong correlation between the residue's solvent-accessible surface and the fitted model parameters.     

Solvent effect on reaction rates in molten salts

Predominant solvation of activated complex is shown to be characteristic for chemical and electrochemical reactions in molten salts.

---

, .

---

---

Presented at EUCHEM Conference on Molten Salts, September 2–8, 1990, Rio-Patras, Greece