Protein-solvent Interactions Research Articles

Quantifying the Molecular Origins of Opposite Solvent Effects on Protein-Protein Interactions

Although the nature of solvent-protein interactions is generally weak and non-specific, addition of cosolvents such as denaturants and osmolytes strengthens protein-protein interactions for some proteins, whereas it weakens protein-protein interactions for others. This is exemplified by the puzzling observation that addition of glycerol oppositely affects the association constants of two antibodies, D1.3 and D44.1, with lysozyme. To resolve this conundrum, we develop a methodology based on the thermodynamic principles of preferential interaction theory and the quantitative characterization of local protein solvation from molecular dynamics simulations. We find that changes of preferential solvent interactions at the protein-protein interface quantitatively account for the opposite effects of glycerol on the antibody-antigen association constants. Detailed characterization of local protein solvation in the free and associated protein states reveals how opposite solvent effects on protein-protein interactions depend on the extent of dewetting of the protein-protein contact region and on structural changes that alter cooperative solvent-protein interactions at the periphery of the protein-protein interface. These results demonstrate the direct relationship between macroscopic solvent effects on protein-protein interactions and atom-scale solvent-protein interactions, and establish a general methodology for predicting and understanding solvent effects on protein-protein interactions in diverse biological environments.

Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration.

Polar Solvents Decrease the Viscosity of High Concentration IgG1 Solutions Through Hydrophobic Solvation and Interaction: Formulation and Biocompatibility Considerations

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Γ23 ) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein-solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1182-1193, 2013.

Hydration shells with a pinch of salt

The discovery of extreme halophile microorganisms in the Dead Sea, which are specifically dependent on a multimolar salt environment to survive, stimulated major developments in biology and physical chemistry. The minireview focuses on the molecular level. After a brief introduction to the history of halophile studies, protein and nucleic acid solvent interactions and their influence on macromolecular structure stabilization and dynamics are discussed.

Molecular Dynamics Simulations of Gammas-Crystallin

Cataract, a leading cause of blindness worldwide, is caused by a loss of solubility of the eye lens structural proteins, called crystallins, that leads to spatial fluctuations in density and the formation of aggregates that scatter light. gammaS-crystallin is the most abundant structural protein in the human eye lens cortex and has been linked to an autosomal dominant early-onset cataract. It consists of two double Greek key domains which, although not identical in sequence, are highly symmetric, making this protein essentially a structural homodimer. Previously (Brubaker et al. Biophys. J. (2011) 100:498), we have shown that although the cataract-related G18V variant aggregates more readily than either wild-type or the symmetry-related G106V variant, it is not the most strongly destabilized. NMR structural studies of human gammaS wild-type and G18V revealed that the cataract-related variant remains fully folded and monomeric under experimental conditions with local structural changes around the point mutation (Brubaker and Martin, Biomol. NMR Assign. (2012) 6:63). These results suggest that aggregation propensity in gammaS-crystallin is not mediated purely by thermodynamic stability, but by a more complex interplay of protein-protein and solvent-protein interactions. Here, we report on atomistic MD simulations using recently determined human gammaS NMR structures of the wild-type and G18V variant as initial configurations to gain detailed insight into the changes in structural and hydration dynamics that result from cataract-related modifications in protein sequence.

Characterization and optimization of a novel protein refolding methodology

Controlling the refolding process of proteins is of considerable theoretical and practical interest [1, 2]. Recently, a simple yet effective method to recover refolded and active proteins has been developed, based on the association of an anionic detergent (the sodium dodecyl sulfate, SDS, known as denaturing agent) with an amphipathic diol solvent [3–5]. More precisely, it has been shown that peculiar solvents can modulate the denaturing properties of SDS and even induce the refolding from a SDS-denatured state. Up to now, this cosolvent effect has been observed on both soluble and bacterial membrane proteins. None to say, it is crucial to have a clear picture of the physicochemical and molecular basis of these refolding processes. To this end, we have used both experimental (intrinsic fluorescence and circular dichroism) and theoretical (molecular dynamics [6]) approaches to help understanding such intricate phenomena including the complex interplay between protein-SDS, protein-solvent and solvent-SDS interactions. In that context, a fundamental investigation has been achieved starting from the study of the detergent/cosolvent couple and then considering the protein/detergent/cosolvent complex. In a first stage, we explore the micellization characteristics of SDS in presence (or absence) of solvents (chain length, one or several alcohols groups, position isomers, ... ). Such a study gives access to the nature and strength of the interactions between the molecules, the system stability as a function of time, and the micelles formation kinetics. Original detergent/solvent pairs can be proposed in order to improve the efficiency of our refolding protocol, as well as to sharpen our understanding of its mechanism, paving the way to the full protein (especially of tricky systems as membrane proteins) characterization.

Water-Protein Interactions: The Secret of Protein Dynamics

Water-protein interactions help to maintain flexible conformation conditions which are required for multifunctional protein recognition processes. The intimate relationship between the protein surface and hydration water can be analyzed by studying experimental water properties measured in protein systems in solution. In particular, proteins in solution modify the structure and the dynamics of the bulk water at the solute-solvent interface. The ordering effects of proteins on hydration water are extended for several angstroms. In this paper we propose a method for analyzing the dynamical properties of the water molecules present in the hydration shells of proteins. The approach is based on the analysis of the effects of protein-solvent interactions on water protons NMR relaxation parameters. NMR relaxation parameters, especially the nonselective (R 1 NS ) and selective (R 1 SE ) spin-lattice relaxation rates of water protons, are useful for investigating the solvent dynamics at the macromolecule-solvent interfaces as well as the perturbation effects caused by the water-macromolecule interactions on the solvent dynamical properties. In this paper we demonstrate that Nuclear Magnetic Resonance Spectroscopy can be used to determine the dynamical contributions of proteins to the water molecules belonging to their hydration shells.

Molecular Dynamics Simulations Provide Atomistic Insight into Hydrogen Exchange Mass Spectrometry Experiments

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.

Understanding the Dissolution of α-Zein in Aqueous Ethanol and Acetic Acid Solutions

Zein is a corn prolamin that has broad industrial applications because of its unique physical properties. Currently, the high cost of extraction and purification, which is directly related to the dispersion of zein in different solvents, is the major bottleneck of the zein industry. Solution behaviors of zein have been studied for a long time. However, the physical nature of zein in different solvents remains unclear. In this study, small-angle X-ray scattering (SAXS), static light scattering (SLS), and rheology were combined to study the structure and protein-solvent interaction of α-zein in both acetic acid and aqueous ethanol solutions. We found that the like-dissolve-like rule, the partial unfolding, and the protonation of zein are all critical to understanding the solution behaviors. Zein holds an elongated conformation (i.e., prolate ellipsoid) in all solutions, as revealed from SAXS data. There is an "aging effect" for zein in aqueous ethanol solutions, as evidenced by the transition of Newtonian rheological profiles for fresh zein solutions to the non-Newtonian shear thinning behavior for zein solutions after storage at room temperature for 24 h. Such shear thinning behavior becomes more pronounced for zein solutions at higher concentrations. The SLS results clearly show that acetic acid is a better solvent to dissolve zein than aqueous ethanol solution, as supported by a more negative second virial coefficient. This is majorly caused by the protonation of the protein, which was further verified by the dissolution of zein in water (a nonsolvent for zein) with the addition of acids.

The Reversible Opening of Water Channels in Cytochrome c Modulates the Heme Iron Reduction Potential

Dynamic protein-solvent interactions are fundamental for life processes, but their investigation is still experimentally very demanding. Molecular dynamics simulations up to hundreds of nanoseconds can bring to light unexpected events even for extensively studied biomolecules. This paper reports a combined computational/experimental approach that reveals the reversible opening of two distinct fluctuating cavities in Saccharomyces cerevisiae iso-1-cytochrome c. Both channels allow water access to the heme center. By means of a mixed quantum mechanics/molecular dynamics (QM/MD) theoretical approach, the perturbed matrix method (PMM), that allows to reach long simulation times, changes in the reduction potential of the heme Fe(3+)/Fe(2+) couple induced by the opening of each cavity are calculated. Shifts of the reduction potential upon changes in the hydration of the heme propionates are observed. These variations are relatively small but significant and could therefore represent a tool developed by cytochrome c for the solvent driven, fine-tuning of its redox functionality.

Structural analysis of Ca2+ dependent and Ca2+ independent type II antifreeze proteins: A comparative molecular dynamics simulation study

Comparative molecular dynamics simulations of Ca2+ dependent psychrophilic type II antifreeze protein (AFP) from herring (Clupea harengus) (hAFP) and Ca2+ independent type II antifreeze protein from long snout poacher (Brachyopsis rostratus) (lpAFP) have been performed for 10ns each at five different temperatures. We have tried to investigate whether the Ca2+ dependent protein obtains any advantage in nature over the independent one. To this end the dynamic properties of these two proteins have been compared in terms of secondary structure content, molecular flexibility, solvent accessibility, intra molecular hydrogen bonds and protein–solvent interactions. At 298 and 373K the flexibility of the Ca2+ independent molecule is higher which indicates that Ca2+ could contribute to stabilize the structure. The thermal unfolding pathways of the two proteins have also been monitored. The rate of unfolding is similar up to 373K, beyond that hAFP shows faster unfolding than lpAFP. The essential subspaces explored by the simulations of hAFP and lpAFP at different temperatures are significantly different as revealed from principal component analysis. Our results may help in understanding the role of Ca2+ for hAFP to express antifreeze activity. Furthermore our study may also help in elucidating the molecular basis of thermostability of two structurally similar proteins, which perform the same function in different manner, one in presence of Ca2+, and the other in absence of the same.

Parameterization of solvent–protein interaction and its use on NMR protein structure determination

NMR structure determination is frequently hindered by an insufficient amount of distance information for determining the correct fold of the protein in its early stages. In response we introduce a simple and general structure-based metric that can be used to incorporate NMR-based restraints on protein surface accessibility. This metric is inversely proportional to the sum of the inverse square distances to neighboring heavy atoms. We demonstrate the use of this restraint using a dataset from the water to protein magnetization transfer experiment on the protein Bax and the solvent paramagnetic relaxation enhancement experiment on the protein ubiquitin and Qua1 homodimer. The calculated solvent accessibility values using the new empirical function are well correlated with the experimental data. By incorporating an associated energy term into Xplor-NIH, we show that structure calculation with a limited number of additional experimental restraints, improves both the precision and accuracy of the resulting structures. This new empirical energy term will have general applicability to other types of solvent accessibility data.

Formyl‐coenzyme A (CoA):oxalate CoA‐transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability

Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a "speckled" charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein-solvent interactions.

Ion specific influences on the stability and unfolding transitions of a naturally aggregating protein; RecA

The Escherichia coli RecA protein is a naturally aggregated protein complex that is affected by the presence of salts. In order to gain further insight into the nature of the ion-interactions on a naturally aggregating protein we used circular dichroism (CD), fluorescence and dynamic light scattering (DLS) to study the effects of different concentrations of MgCl2, CaCl2, NaCl, Na2SO4, and MgSO4 on RecA structure and thermal unfolding. The results show unique ion influences on RecA structure, aggregation, unfolding transitions and stability and the anion effects correlate with the reverse Hofmeister series. The mechanisms of the ion-induced changes most likely result from specific ion binding, changes in the interfacial tension and altered protein–solvent interactions that may be especially important for protein–protein interactions in naturally aggregating proteins. The presence of some ions leads to the formation of RecA complexes that are resistant to complete denaturation and nonspecific aggregation.

Temperature and solvent dependence of the dynamical landscape of tau protein conformations.

We report the variation with temperature of the ensemble distribution of conformations spanned by the tau protein in its dynamical states measured by small-angle X-ray scattering (SAXS) using synchrotron radiation. The SAXS data show a clear temperature variation of the distribution of occupied protein conformations from 293 to 318K. More conformations with a smaller radius of gyration are occupied at higher temperature. The protein-solvent interactions are shown by computer simulation to be essential for controlling the dynamics of protein conformations, providing evidence for the key role of water solvent in the protein dynamics, as proposed by Giorgio Careri.

Bacterial Osmoregulation: A Paradigm for the Study of Cellular Homeostasis

To thrive, cells must control their own physical and chemical properties. This process is known as cellular homeostasis. The dilute solutions traditionally favored by experimenters do not simulate the cytoplasm, where macromolecular crowding and preferential interactions among constituents may dominate critical processes. Solutions that do simulate cytoplasmic conditions are now being characterized. Corresponding cytoplasmic properties can be varied systematically by imposing osmotic stress. This osmotic stress approach is revealing how cytoplasmic properties modulate protein folding and protein?nucleic acid interactions. Results suggest that cytoplasmic homeostasis may require adjustments to multiple, interwoven cytoplasmic properties. Osmosensory transporters with diverse structures and bioenergetic mechanisms activate in response to osmotic stress as other proteins inactivate. These transporters are serving as paradigms for the study of in vivo protein-solvent interactions. Experimenters have proposed three different osmosensory mechanisms. Distinct mechanisms may exist, or these proposals may reflect different perceptions of a single, unifying mechanism.

Reexamining protein–protein and protein–solvent interactions from Kirkwood-Buff analysis of light scattering in multi-component solutions

The classic analysis of Rayleigh light scattering (LS) is re-examined for multi-component protein solutions, within the context of Kirkwood-Buff (KB) theory as well as a more generalized canonical treatment. Significant differences arise when traditional treatments that approximate constant pressure and neglect concentration fluctuations in one or more (co)solvent/co-solute species are compared with more rigorous treatments at constant volume and with all species free to fluctuate. For dilute solutions, it is shown that LS can be used to rigorously and unambiguously obtain values for the osmotic second virial coefficient (B(22)), in contrast with recent arguments regarding protein interactions deduced from LS experiments. For more concentrated solutions, it is shown that conventional analysis over(under)-estimates the magnitude of B(22) for significantly repulsive(attractive) conditions, and that protein-protein KB integrals (G(22)) are the more relevant quantity obtainable from LS. Published data for α-chymotrypsinogen A and a series of monoclonal antibodies at different pH and salt concentrations are re-analyzed using traditional and new treatments. The results illustrate that while traditional analysis may be sufficient if one is interested in only the sign of B(22) or G(22), the quantitative values can be significantly in error. A simple approach is illustrated for determining whether protein concentration (c(2)) is sufficiently dilute for B(22) to apply, and for correcting B(22) values from traditional LS regression at higher c(2) values. The apparent molecular weight M(2, app) obtained from LS is shown to generally not be equal to the true molecular weight, with the differences arising from a combination of protein-solute and protein-cosolute interactions that may, in principle, also be determined from LS.

Backbone and Side-Chain Contributions in Protein Denaturation by Urea

Urea is a commonly used protein denaturant, and it is of great interest to determine its interaction with various protein groups to elucidate the molecular basis of its effect on protein stability. Using the Trp-cage miniprotein as a model system, we report what we believe to be the first computation of changes in the preferential interaction coefficient of the protein upon urea denaturation from molecular-dynamics simulations and examine the contributions from the backbone and the side-chain groups. The preferential interaction is obtained from reversible folding/unfolding replica exchange molecular-dynamics simulations of Trp-cage in presence of urea, over a wide range of urea concentration. The increase in preferential interaction upon unfolding is dominated by the side-chain contribution, rather than the backbone. Similar trends are observed in simulations using two different force fields, Amber94 and Amber99sb, for the protein. The magnitudes of the side-chain and backbone contributions differ in the two force fields, despite containing identical protein-solvent interaction terms. The differences arise from the unfolded ensembles sampled, with Amber99sb favoring conformations with larger surface area and lower helical content. These results emphasize the importance of the side-chain interactions with urea in protein denaturation, and highlight the dependence of the computed driving forces on the unfolded ensemble sampled.

Crystallization and preliminary X-ray crystallographic analysis of a thermostable organic solvent-tolerant lipase from Bacillus sp. strain 42.

An organic solvent-tolerant lipase from Bacillus sp. strain 42 was crystallized using the capillary-tube method. The purpose of studying this enzyme was in order to better understand its folding and to characterize its properties in organic solvents. By initially solving its structure in the native state, further studies on protein-solvent interactions could be performed. X-ray data were collected at 2.0 Å resolution using an in-house diffractometer. The estimated crystal dimensions were 0.09×0.19×0.08 mm. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=117.41, b=80.85, c=99.44 Å, β=96.40°.

Solvent properties governing protein partitioning in polymer/polymer aqueous two-phase systems

Distribution coefficients of various proteins were measured in aqueous Dextran–Ficoll, Dextran–PES, and Ficoll–PES two-phase systems, containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. The acquired data were combined with data for the same proteins in different systems reported previously [29,30] and known solvatochromic solvent properties of the systems [17] to characterize the protein–solvent interactions. The relative susceptibilities of proteins to solvent dipolarity/polarizability, solvent hydrogen bond acidity, solvent hydrogen bond basicity, and solvent ability to participate in ion–ion and ion–dipole interactions were characterized. These parameters, which are representative of solute–solvent interactions, adequately described the partitioning of the proteins in each system. It was found that the relative susceptibilities of proteins to solvent dipolarity/polarizability are interrelated with their relative susceptibilities to solvent hydrogen bond acidity and solvent hydrogen bond basicity similarly to those established previously for small nonionic organic compounds.