Mesophilic Pyrophosphatase Function at High Temperature: A Molecular Dynamics Simulation Study.

The mesophilic inorganic pyrophosphatase from Escherichia coli (EcPPase) retains function at 353 K, the physiological temperature of hyperthermophilic Thermococcus thioreducens, whereas the homolog protein (TtPPase) from this hyperthermophilic organism cannot function at room temperature. To explain this asymmetric behavior, we examined structural and dynamical properties of the two proteins using molecular dynamics simulations. The global flexibility of TtPPase is significantly higher than its mesophilic homolog at all tested temperature/pressure conditions. However, at 353 K, EcPPase reduces its solvent-exposed surface area and increases subunit compaction while maintaining flexibility in its catalytic pocket. In contrast, TtPPase lacks this adaptability and has increased rigidity and reduced protein/water interactions in its catalytic pocket at room temperature, providing a plausible explanation for its inactivity near room temperature.

Effects of pressure on the dynamics of an oligomeric protein from deep-sea hyperthermophile.

Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from a hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is up to 100 MPa (1 kbar). It has attracted great interest in biophysical research because of its high activity under extreme conditions in the seabed. In this study, we use the quasielastic neutron scattering (QENS) technique to investigate the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range. The ß-relaxation dynamics of proteins was studied in the time ranges from 2 to 25 ps, and from 100 ps to 2 ns, using two spectrometers. Our results indicate that, under a pressure of 100 MPa, close to that of the native environment deep under the sea, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), at all measured temperatures, opposite to what we observed previously under ambient pressure. This contradictory observation provides evidence that the protein energy landscape is distorted by high pressure, which is significantly different for hyperthermophilic (IPPase) and mesophilic (HEWL) proteins. We further derive from our observations a schematic denaturation phase diagram together with energy landscapes for the two very different proteins, which can be used as a general picture to understand the dynamical properties of thermophilic proteins under pressure.

Experimental Evidence for the Role of Dynamics in pH-Dependent Enzymatic Activity.

Enzymatic activity is heavily influenced by pH, but the rationale for the dynamical mechanism of pH-dependent enzymatic activity has not been fully understood. In this work, combined neutron scattering techniques, including quasielastic neutron scattering (QENS) and small angle neutron scattering (SANS), are used to study the structural and dynamic changes of a model enzyme, xylanase, under different pH and temperature environments. The QENS results reveal that xylanase at optimal pH exhibits faster relaxational dynamics and a lower energy barrier between conformational substates. The SANS results demonstrate that pH affects both xylanase's stability and monodispersity. Our findings indicate that enzymes have optimized stability and function under their optimal pH conditions, with both structure and dynamics being affected. The current study offers valuable insights into enzymatic functionality mechanisms, allowing for broad industrial applications.

Water-protein dynamic coupling and new opportunities for probing it at low to physiological temperatures in aqueous solutions.

Both the structure and dynamics of biomolecules are known to be essential for their biological function. In the dehydrated state, the function of biomolecules, such as proteins, is severely impeded, so hydration is required for bioactivity. The dynamics of the hydrated biomolecules and their hydration water are related - but how closely? The problem involves several layers of complexity. Even for water in the bulk state, the contribution from various dynamic components to the overall dynamics is not fully understood. In biological systems, the effects of confinement on the hydration water further complicate the picture. Even if the various components of the hydration water dynamics are properly understood, which of them are coupled to the protein dynamics, and how? The studies of protein dynamics over the wide temperature range, from physiological to low temperatures, provide some answers to these question. At low temperatures, both the protein and its hydration water behave as solids, with only vibrational degrees of freedom. As the temperature is increased, non-vibrational dynamic components start contributing to the measurable dynamics and eventually become dominant at physiological temperatures. Thus, the temperature dependence of the dynamics of protein and its hydration water may allow probing various dynamic components separately. In order to suppress the water freezing, the low-temperature studies of protein rely on either low-hydrated samples (essentially, hydrated protein powders), or cryo-protective solutions. Both approaches introduce the hydration environments not characteristic of the protein environments in living systems, which are typically aqueous protein solutions of various concentrations. In this paper, we discuss the coupling between the dynamic components of the protein and its hydration water by critical examining of the existing literature, and then propose that proteins can be studied in an aqueous solution that is remarkably similar in its dynamic properties to pure water, yet does not freeze down to about 200 K, even in the bulk form. The first experiment of this kind using quasielastic neutron scattering is discussed, and more experiments are proposed.

Investigations of structural and dynamical mechanisms of ice formation regulated by graphene oxide nanosheets.

Recent research indicates that graphene oxide (GO) nanosheets can be used to regulate ice formation by controlling critical ice nucleus growth in water at supercooling temperatures. In addition, the study of ice formation mechanisms regulated by GO nanosheets, a good model system for antifreeze proteins (AFPs), will shed light on how AFPs regulate ice formation in nature. In this work, time-resolved small-angle x-ray scattering (TR-SAXS) and quasi-elastic neutron scattering (QENS) experiments were carried out to investigate the structural and dynamical mechanisms of ice formation regulated by GO nanosheets. Strikingly, a transient intermediate state was observed in TR-SAXS experiments that only exists in the aqueous dispersions with a larger GO size (11 nm). This serves as evidence that the size of GO is critical for regulating ice formation. Elastic neutron scattering results indicate that ice is formed in all samples and thermal hysteresis occurs in GO aqueous dispersions in both H2O and D2O. The structural and dynamics information about water molecules in GO, extracted from QENS, reveals different dynamical behaviors of water molecules in GO aqueous dispersions when approaching the ice formation temperature.

Proteins remain soft at lower temperatures under pressure.

The low-temperature behavior of proteins under high pressure is not as extensively investigated as that at ambient pressure. In this paper, we study the dynamics of a hydrated protein under moderately high pressures at low temperatures using the quasielastic neutron scattering method. We show that when applying pressure to the protein-water system, the dynamics of the protein hydration water does not slow down but becomes faster instead. The degree of "softness" of the protein, which is intimately related to the enzymatic activity of the protein, shows the same trend as its hydration water as a function of temperature at different pressures. These two results taken together suggest that at lower temperatures, the protein remains soft and active under pressure.

Observation of a dynamic crossover in RNA hydration water which triggers a dynamic transition in the biopolymer.

High-resolution quasielastic neutron scattering spectroscopy was used to measure H2O and D2O hydrated RNA samples. The contribution of scattering from RNA was subtracted out by taking the difference of the signals between the two samples. The measurements were made at a series of temperatures from 270 K down to 180 K. The relaxing-cage model was used to analyze the difference quasielastic spectra. We observed clear evidence of a fragile-to-strong dynamic crossover (FSC) at TL=220 K in RNA hydration water. We further show that the mean-square displacements of the hydrogen atoms in both RNA and its hydration water exhibit a sharp change in slope at approximately the same temperature 220 K. This latter fact suggests that the dynamic transition in RNA is triggered by the abrupt change of mobility of the hydration water at its FSC temperature.

Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation.

Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared with alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure, rhodopsin is swollen by the penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.

Neutron scattering in the biological sciences: progress and prospects.

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.

Collective Excitations in Protein as a Measure of Balance Between its Softness and Rigidity.

In this article, we elucidate the protein activity from the perspective of protein softness and flexibility by studying the collective phonon-like excitations in a globular protein, human serum albumin (HSA), and taking advantage of the state-of-the-art inelastic X-ray scattering (IXS) technique. Such excitations demonstrate that the protein becomes softer upon thermal denaturation due to disruption of weak noncovalent bonds. On the other hand, no significant change in the local excitations is detected in ligand- (drugs) bound HSA compared to the ligand-free HSA. Our results clearly suggest that the protein conformational flexibility and rigidity are balanced by the native protein structure for biological activity.

Enhanced Dynamics of Hydrated tRNA on Nanodiamond Surfaces: A Combined Neutron Scattering and MD Simulation Study.

Nontoxic, biocompatible nanodiamonds (ND) have recently been implemented in rational, systematic design of optimal therapeutic use in nanomedicines. However, hydrophilicity of the ND surface strongly influences structure and dynamics of biomolecules that restrict in situ applications of ND. Therefore, fundamental understanding of the impact of hydrophilic ND surface on biomolecules at the molecular level is essential. For tRNA, we observe an enhancement of dynamical behavior in the presence of ND contrary to generally observed slow motion at strongly interacting interfaces. We took advantage of neutron scattering experiments and computer simulations to demonstrate this atypical faster dynamics of tRNA on ND surface. The strong attractive interactions between ND, tRNA, and water give rise to unlike dynamical behavior and structural changes of tRNA in front of ND compared to without ND. Our new findings may provide new design principles for safer, improved drug delivery platforms.

Quasi-elastic Neutron Scattering Reveals Ligand-Induced Protein Dynamics of a G-Protein-Coupled Receptor.

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond-nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differences can be attributed to the influence of the covalently bound retinal ligand. Furthermore, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.

Temperature-dependent dynamics of dry and hydrated ß-casein studied by quasielastic neutron scattering.

ß-Casein is a component of casein micelle with amphillic nature and is recognized as a "natively disordered" protein that lacks secondary structures. In this study, the temperature and hydration effects on the dynamics of ß-casein are explored by quasielastic neutron scattering (QENS). An upturn in the mean square displacement (MSD) of hydrated ß-casein indicates an increase of protein flexibility at a temperature of ~225 K. Another increase in MSD at ~100 K, observed in both dry and hydrated ß-casein, is ascribed to the methyl group rotations, which are not sensitive to hydration. QENS analysis in the energy domain reveals that the fraction of hydrogen atoms participating in motion in a sphere of diffusion is highly hydration dependent and increases with temperature. In the time domain analysis, a logarithmic-like decay is observed in the range of picosecond to nanosecond (ß-relaxation time) in the dynamics of hydrated ß-casein. This dynamical behavior has been observed in hydrated globular and oligomeric proteins. Our temperature-dependent QENS experiments provide evidence that lack of a secondary structure in ß-casein results in higher flexibility in its dynamics and easier reversible thermal unfolding compared to other rigid biomolecules.

Temperature Dependence of Logarithmic-like Relaxational Dynamics of Hydrated tRNA.

The dynamics of RNA within the ß-relaxation region of 10 ps to 1 ns is crucial to its biological function. Because of its simpler chemical building blocks and the lack of the side methyl groups, faster relaxational dynamics of RNA compared to proteins can be expected. However, the situation is actually opposite. In this work, the relaxational dynamics of tRNA is measured by quasielastic neutron scattering and analyzed using the mode coupling theory, originally developed for glass-forming liquids. Our results reveal that the dynamics of tRNA follows a log-decay within the ß-relaxation region, which is an important trait demonstrated by the dynamics of proteins. The dynamics of hydrated tRNA and lysozyme compared in the time domain further demonstrate that the slower dynamics of tRNA relative to proteins originates from the difference in the folded states of tRNA and proteins, as well as the influence of their hydration water.

Apparent Decoupling of the Dynamics of a Protein from the Dynamics of its Aqueous Solvent.

Studies of the low-temperature dynamics of proteins in aqueous solutions are limited by the crystallization of water. In this work, we use a solution of LiCl in D2O as a solvent for a protein to prevent crystallization and study the dynamics of both the protein and its aqueous solvent by quasielastic neutron scattering (QENS) in the temperature range of 210 to 290 K. Our results reveal that, while the dynamics of the aqueous solvent undergoes a crossover at about 220 K, the dynamics of the protein itself shows no transition at this temperature. The prevailing view is that the ß-fluctuations of the protein are governed by the α-fluctuations of the solvent; therefore, observation of the apparent decoupling between the dynamics of the protein and its solvent below the crossover temperature is remarkable.

Dynamic behavior of oligomeric inorganic pyrophosphatase explored by quasielastic neutron scattering.

The purpose of this investigation is to determine whether a large oligomeric protein, inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens with quaternary structural complexity, would have distinguishable dynamic characteristics compared to those of the small simple monomeric model protein, lysozyme. In this study, the ß-relaxational dynamics of the two proteins, IPPase and lysozyme, are compared in the 10 ps to 0.5 ns time interval using quasi-elastic neutron scattering (QENS). Both of the protein dynamics show a characteristic logarithmic-like decay in the intermediate scattering function (ISF) of the hydrogen atoms. Distinguishable dynamical behavior found between two proteins reveals local flexibility and conformational substates unique to oligomeric structures. Moreover, the temperature dependence of the mean square displacement (MSD) of the hydrogen atoms in protein molecules, which is a traditional way to determine the "softness" of the protein molecule, is measured and shows no difference for the two proteins.

Diffusion processes in water on oxide surfaces: quasielastic neutron scattering study of hydration water in rutile nanopowder.

Quasielastic neutron scattering (QENS) was used to investigate the diffusion dynamics of hydration water on the surface of rutile (TiO(2)) nanopowder. The dynamics measurements utilizing two inelastic instruments, a backscattering spectrometer and a disk chopper spectrometer, probed the fast, intermediate, and slow motions of the water molecules on the time scale of picoseconds to more than a nanosecond. We employed a model-independent analysis of the data collected at each value of the scattering momentum transfer to investigate the temperature dependence of several diffusion components. All of the probed components were present in the studied temperature range of 230-320 K, providing, at a first sight, no evidence of discontinuity in the hydration water dynamics. However, a qualitative change in the elastic scattering between 240 and 250 K suggested a surface freezing-melting transition, when the motions that were localized at lower temperatures became delocalized at higher temperatures. On the basis of our previous molecular dynamics simulations of this system, we argue that interpretation of QENS data from such a complex interfacial system requires at least qualitative input from simulations, particularly when comparing results from spectrometers with very different energy resolutions and dynamic ranges.

Low-temperature dynamics of water confined in a hydrophobic mesoporous material.

Quasielastic neutron scattering was used to study the dynamics of three-dimensional confined water in a hydrophobic mesoporous material designated as CMK-1 in the temperature range from 250 to 170 K. We observe a crossover phenomenon at temperature T(L) . We find that T(L) of water confined in CMK-1 occurs in between previous observations of one-dimensional confined water in materials with different hydrophilicities. This provides the first evidence that besides the obvious surface effect brought about by the hydrophobic confinements, T(L) is also dependent on the dimensionality of the geometry of the confinement.

Studies of phononlike low-energy excitations of protein molecules by inelastic x-ray scattering.

Molecular dynamics simulations and neutron scattering experiments have shown that many hydrated globular proteins exhibit a universal dynamic transition at TD = 220 K, below which the biological activity of a protein sharply diminishes. We studied the phononlike low-energy excitations of two structurally very different proteins, lysozyme and bovine serum albumin, using inelastic x-ray scattering above and below TD. We found that the excitation energies of the high-Q phonons show a marked softening above TD. This suggests that the large amplitude motions of wavelengths corresponding to this specific Q range are intimately correlated with the increase of biological activities of the proteins.