Protein Hydration Shell Research Articles

PELDOR/DEER: An Electron Paramagnetic Resonance Method to Study Membrane Proteins in Lipid Bilayers.

Every membrane protein is involved in close interactions with the lipid environment of cellular membranes. The annular lipids, that are in direct contact with the polypeptide, can in principle be seen as an integral part of its structure, akin to the first hydration shell of soluble proteins. It is therefore desirable to investigate the structure of membrane proteins and especially their conformational flexibility under conditions that are as close as possible to their native state. Thiscan beachieved by reconstituting the protein into proteoliposomes, nanodiscs, or bicelles. In recent years, PELDOR/DEER spectroscopy has proved to be a very useful method to study the structure and function of membrane proteins in such artificial membrane environments. The technique complementsboth X-ray crystallography andcryo-EM and can be used in combination with virtually any artificial membrane environment and under certain circumstances even in native membranes. Of the above-mentioned membrane mimics, bicelles are currently the least often used for PELDOR studies, although they offer some advantages, especially their ease of use. Here, we provide a step-by-step protocol for studying a bicelle reconstituted membrane protein with PELDOR/DEER spectroscopy.

Mobility of Water, Urea and Trimethylamine-N-Oxide Molecules in the Vicinity of Globular Protein

Molecular mobility in the hydration shell of the SNase globular protein in an aqueous solution with cosolvents (urea and trimethylamine oxide) is studied using all-atom molecular dynamic simulations. Average displacements of the molecules initially located in the successive layers around the protein are calculated over the same short period of time to characterize the diffusion mobility of the molecules depending on the distance to the protein. It is shown that solvent molecules have lower mobility near the protein, and the mobility of more distant molecules increases irregularly and correlates with the positions of the distribution function maxima of these molecules around the protein. After the second maximum of these functions, the mobility reaches its bulky values both for water and the cosolvent.

Mechanism of Trehalose-Induced Protein Stabilization from Neutron Scattering and Modeling.

The sugar molecule trehalose has been proven to be an excellent stabilizing cosolute for the preservation of biological materials. However, the stabilizing mechanism of trehalose has been much debated during the previous decades, and it is still not fully understood, partly because it has not been completely established how trehalose molecules structure around proteins. Here, we present a molecular model of a protein-water-trehalose system, based on neutron scattering results obtained from neutron diffraction, quasielastic neutron scattering, and different computer modeling techniques. The structural data clearly show how the proteins are preferentially hydrated, and analysis of the dynamical properties show that the protein residues are slowed down because of reduced dynamics of the protein hydration shell, rather than because of direct trehalose-protein interactions. These findings, thereby, strongly support previous models related to the preferential hydration model and contradict other models based on water replacement at the protein surface. Furthermore, the results are important for understanding the specific role of trehalose in biological stabilization and, more generally, for providing a likely mechanism of how cosolutes affect the dynamics of proteins.

Ordering effect of protein surfaces on water dynamics: NMR relaxation study

Proteins in solution affect the structural and dynamic properties of the bulk water at the protein-water interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step toward the determination of the size of the water ordering contributions originate in proteins systems.

Low temperature dependence of protein-water interactions on barstar surface: A nano-scale modelling

The dynamics of the hydration shell of the inhibitor barstar is analysed at low temperature (300–243 K), through all-atom molecular dynamics simulations, and compared with that of bulk water. The relaxation of residence times of solvent molecules in the protein hydration shell follows a stretched exponential function exp[−(t/τ)β] with β = 0.48 ± 0.01, independent of temperature, showing that the decay process is mainly dominated by long-range molecular relaxation channels (short-range for bulk water). The percentage of water molecules exhibiting 4 hydrogen bonds, xHB4, is found to be a parameter essential for understanding some room and low temperature dependent properties of the protein hydration shell, suggesting an explanation for the unfreezing of protein hydration water as temperature decrease below 273 K. Moreover the dynamical transition that proteins and their hydration water exhibit at ~225 K can be explained by the decrease of ‘hydrogen bond defects’ in the protein hydration shell as temperature goes down. If most of those water molecules would present a tetrahedral arrangement (nearly no ‘hydrogen bond defects’), the bioactivity of proteins would be negligible. Comparison with experimental results is provided all along the work. Experimental data are quantitatively reproduced.

Dipolar susceptibility of protein hydration shells

We present a formalism for the calculation of interfacial dielectric constant of hydration shells of proteins from configurations produced by atomistic numerical simulations. The theoretical approach is applied to classical molecular dynamics simulations of hydrated cytochrome c protein in the range of temperatures from 280 to 330 K. The interfacial dielectric constant was found to be equal to 2–4 depending on temperature. This dielectric constant reflects constraints imposed by the protein on the hydration waters and their low ability to polarize in response to an external field.

Sugar-Mediated Stabilization of Protein against Chemical or Thermal Denaturation.

The protective action of sugars against the denaturation of myoglobin was clarified by X-ray and neutron scattering methods. Different types of sugars such as disaccharides (trehalose, sucrose) and monosaccharides (glucose, fructose) were used. Experimental data and theoretical simulation based on three different solvation models (preferential solvation model, nonpreferential solvation model, and preferential exclusion (hydration) model) indicated that sugar molecules were preferentially or weakly excluded from the protein surface and preserved the native protein hydration shell. This trend was more evident for disaccharides. The preferential exclusion shifted gradually to the nonpreferential solvation at higher sugar concentrations. On the protective actions of the sugars against the guanidinium-chloride-mediated denaturation, all sugars, starting from the low concentration of 5% w/v, showed the protective trend toward the protein native structure, especially for the secondary structure. The thermal structural transition temperature of myoglobin was raised by about 4-5 °C, accompanied by amyloid formation, for all hierarchical structural levels. In particular, the oligomer formation of the amyloid aggregates was more suppressed. The above protective action was sugar-dependent. The present results clearly suggest that sugars intrinsically protect the native structure of proteins against chemical and thermal denaturation through the preservative action of the hydration shell.

In-Cell Titration of Small Solutes Controls Protein Stability and Aggregation.

The components of the intracellular environment vary widely in size: from large multiprotein complexes to atomic ions. Besides water, low-molecular-weight solutes (<1 kDa) such as electrolytes, metabolites, and carbohydrates are by far the most abundant of these components. Small solutes are thus key contributors to the solvation environment in the cell. Small solutes have been known for decades to alter protein structure or activity in vitro, through their interactions with protein surfaces or hydration shells. Here we use the cell itself as our test tube, by titrating its hydration, ion, or carbohydrate composition systematically. We trigger the selective uptake of specific solutes by exposing cells to hyperosmotic media. We then measure protein structure, stability, unfolding kinetics, and aggregation in these different intracellular environments by using fast relaxation imaging. We compare these results with controls where solutes cannot enter the cell and only hydration is altered. Protein structure, thermal stability, and aggregation onset all depend on the concentration and chemical nature of the solute titrated into the cell. Our work highlights the important contributions of small solutes in defining how proteins interact within the cell and suggests that intracellular variation of the solute composition could be an important regulator of protein function.

Protective action of trehalose and glucose on protein hydration shell clarified by using X-ray and neutron scattering

Sugars are well known to retain protein structures and to protect denaturation. Protective actions of sugars on protein structures have been discussed under issues of preferential hydration and/or interaction between sugar and protein. By using synchrotron radiation wide-angle X-ray scattering (SR-WAXS) and small-angle neutron scattering (SANS) methods, we have succeeded to obtain a direct evidence of the effect of trehalose and glucose on a protein structure and hydration. Myoglobin from horse-skeletal muscles was used as one of typical globular proteins. The sugars used were trehalose and glucose whose concentrations were varied from 0% w/w to 35% w/w. The experimental SR-WAXS and SANS data combined with theoretical scattering function simulation and fitting clearly show that both sugars are preferentially excluded from the hydration-shell region of the protein to preserve hydration-shell density. up to the sugar concentration of ∼25% w/w. Compared with that of glucose (monosaccharide), the preservation action of the protein hydration-shell is much evidently seen for the case of trehalose (disaccharide), which might relate to a role of the preferential production of trehalose by organisms against extreme environmental stress.

Germanium Plasmon Enhanced Resonators for Label-Free Terahertz Protein Sensing

Abstract A Terahertz protein sensing concept based on subwavelength Ge resonators is presented. Ge bowtie resonators, compatible with CMOS fabrication technology, have been designed and characterized with a resonance frequency of 0.5 THz and calculated local intensity enhancement of 10.000. Selective biofunctionalization of Ge resonators on Si wafer was achieved in one step using lipoic acid-HaloTag ligand (LA-HTL) for biofunctionalization and passivation. The results lay the foundation for future investigation of protein tertiary structure and the dynamics of protein hydration shell in response to protein conformation changes.

Structure of Aqueous Solutions of Trimethylaminoxide, Urea, and Their Mixture

Aqueous solutions of natural osmolytes (trimethylaminoxide (TMAO), urea, and their mixture) at relatively small (biologically relevant) concentrations are analyzed by the all-atom molecular dynamics simulation. In the recent work (Smolin N. et al. PCCP. 2017. 19. P. 6345) it has been noted that in the protein hydration shell the fraction of TMAO molecules is much smaller than that of urea. The urea addition causes a further decrease in the TMAO fraction in the protein hydration shell. This work shows that in binary solutions urea fraction at urea molecules is always larger than the bulk urea concentration. At the same time, the TMAO fraction near TMAO is the same as in the bulk. In ternary solutions, TMAO and urea behave the same as the binary ones, i.e. they do not noticeably affect each other. This means that the behavior of TMAO and urea molecules in the protein hydration shell is associated with protein rather than their interaction with each other.

Characterizing Hydration Properties Based on the Orientational Structure of Interfacial Water Molecules.

In this work, we present a general computational method for characterizing the molecular structure of liquid water interfaces as sampled from atomistic simulations. With this method, the interfacial structure is quantified based on the statistical analysis of the orientational configurations of interfacial water molecules. The method can be applied to generate position dependent maps of the hydration properties of heterogeneous surfaces. We present an application to the characterization of surface hydrophobicity, which we use to analyze simulations of a hydrated protein. We demonstrate that this approach is capable of revealing microscopic details of the collective dynamics of a protein hydration shell.

Terahertz absorption of lysozyme in solution.

Absorption of radiation by solution is described by its frequency-dependent dielectric function and can be viewed as a specific application of the dielectric theory of solutions. For ideal solutions, the dielectric boundary-value problem separates the polar response into the polarization of the void in the liquid, created by the solute, and the response of the solute dipole. In the case of a protein as a solute, protein nuclear dynamics do not project on significant fluctuations of the dipole moment in the terahertz domain of frequencies and the protein dipole can be viewed as dynamically frozen. Absorption of radiation then reflects the interfacial polarization. Here we apply an analytical theory and computer simulations to absorption of radiation by an ideal solution of lysozyme. Comparison with the experiment shows that Maxwell electrostatics fails to describe the polarization of the protein-water interface and the "Lorentz void," which does not anticipate polarization of the interface by the external field (no surface charges), better represents the data. An analytical theory for the slope of the solution absorption against the volume fraction of the solute is formulated in terms of the cavity field response function. It is calculated from molecular dynamics simulations in good agreement with the experiment. The protein hydration shell emerges as a separate sub-ensemble, which, collectively, is not described by the standard electrostatics of dielectrics.

Liquefaction of Biopolymers: Solvent-free Liquids and Liquid Crystals from Nucleic Acids and Proteins.

ConspectusBiomacromolecules, such as nucleic acids, proteins, and virus particles, are persistent molecular entities with dimensions that exceed the range of their intermolecular forces hence undergoing degradation by thermally induced bond-scission upon heating. Consequently, for this type of molecule, the absence of a liquid phase can be regarded as a general phenomenon. However, certain advantageous properties usually associated with the liquid state of matter, such as processability, flowability, or molecular mobility, are highly sought-after features for biomacromolecules in a solvent-free environment. Here, we provide an overview over the design principles and synthetic pathways to obtain solvent-free liquids of biomacromolecular architectures approaching the topic from our own perspective of research. We will highlight the milestones in synthesis, including a recently developed general surfactant complexation method applicable to a large variety of biomacromolecules as well as other synthetic principles granting access to electrostatically complexed proteins and DNA.These synthetic pathways retain the function and structure of the biomacromolecules even under extreme, nonphysiological conditions at high temperatures in water-free melts challenging the existing paradigm on the role of hydration in structural biology. Under these conditions, the resulting complexes reveal their true potential for previously unthinkable applications. Moreover, these protocols open a pathway toward the assembly of anisotropic architectures, enabling the formation of solvent-free biomacromolecular thermotropic liquid crystals. These ordered biomaterials exhibit vastly different mechanical properties when compared to the individual building blocks. Beyond the preparative aspects, we will shine light on the unique potential applications and technologies resulting from solvent-free biomacromolecular fluids: From charge transport in dehydrated liquids to DNA electrochromism to biocatalysis in the absence of a protein hydration shell. Moreover, solvent-free biological liquids containing viruses can be used as novel storage and process media serving as a formulation technology for the delivery of highly concentrated bioactive compounds. We are confident that this new class of hybrid biomaterials will fuel further studies and applications of biomacromolecules beyond water and other solvents and in a much broader context than just the traditional physiological conditions.

Solvent-Slaved Dynamic Processes Observed by Tryptophan Phosphorescence of Human Serum Albumin

Despite extensive experimental and computational efforts to understand the nature of the hierarchy of protein fluctuations and the modulating role of the protein hydration shell, a detailed microscopic description of the dynamics of the protein-solvent system has yet to be achieved. By using single tryptophan protein phosphorescence, we follow site-specific internal protein dynamics over a broad temperature range and demonstrate three independent dynamic processes. Process I is seen at temperatures below the bulk solvent Tg, has low activation energy, and is likely due to fast vibrations that may be enabled by water mobility on the protein surface. Process II is observed above 170 K, with activation energy typical of β relaxations in a glass; it has the same temperature dependence as fluctuations of hydration shell waters. Process III is observed at T > 200 K; it has super-Arrhenius temperature dependence and closely follows the primary relaxation of the bulk. The fluorescence of pyranine bound to the protein reports on the mobility of water in the hydration shell; it reveals a shift in emission spectra with increasing temperature, indicative of a changing H-bond network at the surface of the protein. These results support a model of solvent-slaved protein dynamics.

Role of the hydrophobic and hydrophilic sites in the dynamic crossover of the protein-hydration water

Abstract Molecular dynamics simulations were performed to study the water structure and dynamics in the hydration shell of the globular TS-Kappa protein. The results show that for a wide range of temperatures the diffusion coefficient of water near the protein surface is lower than in bulk. A crossover in the diffusion behavior of hydration water is observed at different temperatures for hydrophilic and hydrophobic vicinities. We have found a correlation between the crossover in the hydrophilic case and the protein dynamical transition. An explanation in terms of the competition between water–water water–protein H-bond formation is provided based on H-bond network analysis.

Temperature-mediated switching of protectant-denaturant behavior of trimethylamine-N-oxide and consequences on protein stability from a replica exchange molecular dynamics simulation study

The detailed mechanism of protein folding–unfolding processes with the aid of osmolytes has been a leading topic of discussion over many decades. We have used replica-exchange molecular dynamics simulation to propose the molecular mechanism of interaction of a 20-residue mini-protein with urea and trimethylamine N-oxide (TMAO) that act as denaturing and protecting osmolyte, respectively, in binary osmolyte solutions. Urea is found to exert its action by interacting directly with the protein residues. Temperature tolerance of TMAO’s action is particularly emphasised in this study. At lower range of temperature, TMAO acts as a successful protein protectant. Interestingly, the study discloses the tendency of TMAO molecules to prefer self-association at the protein surface at elevated temperature. A greater number of TMAO molecules in the protein hydration shell at higher temperature is also observed. Dihedral angle principal component analysis and free energy landscape plots sampled all possible conformations adopted by the protein that reveal highly folded behaviour of the protein in pure water and binary TMAO solutions and highly unfolded behaviour in presence of urea.

Accurate optimization of amino acid form factors for computing small-angle X-ray scattering intensity of atomistic protein structures.

Structure modelling via small-angle X-ray scattering (SAXS) data generally requires intensive computations of scattering intensity from any given biomolecular structure, where the accurate evaluation of SAXS profiles using coarse-grained (CG) methods is vital to improve computational efficiency. To date, most CG SAXS computing methods have been based on a single-bead-per-residue approximation but have neglected structural correlations between amino acids. To improve the accuracy of scattering calculations, accurate CG form factors of amino acids are now derived using a rigorous optimization strategy, termed electron-density matching (EDM), to best fit electron-density distributions of protein structures. This EDM method is compared with and tested against other CG SAXS computing methods, and the resulting CG SAXS profiles from EDM agree better with all-atom theoretical SAXS data. By including the protein hydration shell represented by explicit CG water molecules and the correction of protein excluded volume, the developed CG form factors also reproduce the selected experimental SAXS profiles with very small deviations. Taken together, these EDM-derived CG form factors present an accurate and efficient computational approach for SAXS computing, especially when higher molecular details (represented by the q range of the SAXS data) become necessary for effective structure modelling.

Thermal contraction of aqueous glycerol and ethylene glycol solutions for optimized protein-crystal cryoprotection.

The thermal contraction of aqueous cryoprotectant solutions on cooling to cryogenic temperatures is of practical importance in protein cryocrystallography and in biological cryopreservation. In the former case, differential contraction on cooling of protein molecules and their lattice relative to that of the internal and surrounding solvent may lead to crystal damage and the degradation of crystal diffraction properties. Here, the amorphous phase densities of aqueous solutions of glycerol and ethylene glycol at T = 77 K have been determined. Densities with accuracies of <0.5% to concentrations as low as 30%(w/v) were determined by rapidly cooling drops with volumes as small as 70 pl, assessing their optical clarity and measuring their buoyancy in liquid nitrogen-argon solutions. The use of these densities in contraction matching of internal solvent to the available solvent spaces is complicated by several factors, most notably the exclusion of cryoprotectants from protein hydration shells and the expected deviation of the contraction behavior of hydration water from bulk water. The present methods and results will assist in developing rational approaches to cryoprotection and an understanding of solvent behavior in protein crystals.

Proteins in solution: Fractal surfaces in solutions

The concept of the surface of a protein in solution, as well of the interface between protein and 'bulk solution', is introduced. The experimental technique of small angle X-ray and neutron scattering is introduced and described briefly. Molecular dynamics simulation, as an appropriate computational tool for studying the hydration shell of proteins, is also discussed. The concept of protein surfaces with fractal dimensions is elaborated. We finish by exposing an experimental (using small angle X-ray scattering) and a computer simulation case study, which are meant as demonstrations of the possibilities we have at hand for investigating the delicate interfaces that connect (and divide) protein molecules and the neighboring electrolyte solution.