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An improved analytical model for the QENS spectra of proteins is proposed. Dynamics of protein atoms closer to the surface and those closer to the core are separately characterized.

To elucidate, predict, and control the biological functions of proteins, it is important to clarify their solution structure and dynamics. Small-angle scattering is one of effective methods for examining the changes in their structures and dynamics due to their binding with target molecules under physiological conditions, although the spatial resolution of the structure revealed by this method is lower than that by crystallographic technique, which provides structural information at atomic resolution. In addition, neutron spin echo is the only technique that can measure the conformational dynamics of proteins in the Q-region, which is covered by small-angle scattering. In this paper, we will introduce some of our recent studies using small-angle scattering and neutron spin echo.

Incoherent neutron scattering is a powerful tool to investigate the dynamics of biomolecules and water. In this article, a new dynamical model called “Matryoshka model” is first described, which provides detailed information on the motions contained in the phospholipid molecules based on the scattering spectra. In the latter half of the article, a relationship between water activity and water mobility, and the effects of pressure and hydration on protein dynamics are discussed. These studies demonstrate the advancement of analytical methods which provide ever more detailed information for elucidating the mechanism of biological functions.

Epigenetics is an emerging field of science that studies heritable changes caused by activation and deactivation of genes without any change in the genomic DNA information. In other words, epigenetics is the study to elucidate how environment and our behavior can cause changes that affect the way our genes function. Many types of regulatory mechanisms in epigenetics, such as DNA modification and numerous types of histone modification has been reported. Steady state recognition of histone modifications and their localizations on genome has been reported, however, it is still uncertain how the modification is dynamically recognized and contribute for establishing higher order structure in nuclei. In this article, we introduce our recent trials to analyze protein with chemical modifications and/or intrinsic disorder regions by combining biochemistry, organic chemistry and ESR, and also show future plan to utilize neutron scattering to further characterize the relevant proteins.

Quasi-elastic neutron scattering (QENS) is a powerful technique to study protein dynamics. In general, QENS measurements are carried out in D2O solvent whereas functional studies of proteins are conducted in H2O solvent. Therefore, to link the QENS studies with the functional studies and then to understand the molecular basis of protein functions in detail, it is important to investigate the effects of solvent isotopic change on dynamical parameters obtained by QENS. For this purpose, in this study, MD simulations were carried out on hen egg white lysozyme, a well-folded and characterized protein, in H2O and in D2O. The dynamical parameters were extracted from the QENS spectra calculated from the MD trajectories. It was found that isotopic effects depend on energy resolutions and that at the energy resolutions that recent QENS studies often employ, the local dynamical behavior of proteins characterized in D2O more or less reflects that in H2O.

X-ray/neutron fiber diffraction and small-angle X-ray/neutron scattering are widely used to investigate the molecular structure of fibrous proteins, including amyloid fibrils. However, there is sometimes confusion between these two techniques despite the fact that sample conditions and the content of the information obtained are not the same. In this brief chapter, we present the differences in sample conditions between these two methods, and their effects on experimentally obtained diffraction or scattering patterns, emphasizing the degree of disorder in the samples.

Ｘ線や中性子散乱は、溶液中の生体分子の構造状態や集合状態、分子間相互作用などが分かる手法である.放射光Ｘ線では、短時間で高精度な溶液散乱データが得られる.中性子線では重水素化ラベルにより特定の分子を不可視化して、分子集団の中の注目する分子だけを選択的に観測することもできる.また、熱揺らぎと同程度のエネルギーを持つ中性子線では、分子運動の計測にも適している．現在のところ相分離現象の解析への適用は限定的であるが、ナノ～メゾスケールでの相分離状態を解析したり、相分離する過程を追跡したりするのに有効な手法である.

Incoherent neutron scattering (iNS) is one of the most powerful techniques to study the dynamical behavior of bio-macromolecules such as proteins and lipid molecules or whole cells. This technique has widely been used to elucidate the fundamental aspects of molecular motions that manifest in the bio-macromolecules in relation to their intrinsic molecular properties and biological functions. Furthermore, in the last decade, iNS studies focusing on a possible relationship between molecular dynamics and biological malfunctions, i.e., human diseases and disorders, have gained importance. In this review, we summarize recent iNS studies on pathologically relevant proteins and lipids and discuss how the findings are of importance to elucidate the molecular mechanisms of human diseases and disorders that each study targets. Since some diseases such as amyloidosis have become more relevant in the aging society, research in this field will continue to develop further and be more important in the current increasing trend for longevity worldwide.

Clock proteins maintain constant enzymatic activity regardless of temperature, even though thermal fluctuation is accelerated as temperature increases. We investigated temperature influences on the dynamics of KaiC, a temperature-compensated ATPase in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated by a factor of only 1.2 by increasing the temperature by 10°C. This temperature insensitivity of the local motions was not necessarily unique to KaiC, but confirmed also for a series of temperature-sensitive mutants of KaiC and proteins other than clock-related proteins. Rather, the dynamics associated with the temperature-compensatory nature of the reaction- and system-level was found in global diffusional motions, which was suggested to regulate the temperature dependence of ATPase activity and dephosphorylation process presumably through changes in the hexamer conformation of KaiC. The spatiotemporal scale at which cross-scale causality of the temperature sensitivity is established is finite, and extends down to picosecond to sub-nanosecond dynamics only in a very limited part of KaiC, not in its entire part.

Modern phospholipid membranes are known to be in a functional, physiological state, corresponding to the liquid crystalline phase, only under very precise external conditions. The phase is characterised by specific lipid motions, which seem mandatory to permit sufficient flexibility and stability for the membrane. It can be assumed that similar principles hold for proto-membranes at the origin of life although they were likely composed of simpler, single chain fatty acids and alcohols. In the present study we investigated molecular motions of four types of model membranes to shed light on the variations of dynamics and structure as a function of temperature as protocells might have existed close to hot vents. We find a clear hierarchy among the flexibilities of the samples, where some structural parameters seem to depend on the lipids used while others do not.

It is widely accepted that disordered regions in proteins, part of which takes predominantly α-helical conformation to a varying degree, play a critical role in biological function. Structural analysis of these flexible proteins is, however, not straightforward because existing methods to characterize the structural features of the disordered regions are not applicable to all the proteins with various sizes and physicochemical properties. In this study, to gain information on the helical propensity in the disordered regions as well as the overall three-dimensional structures of the proteins, usefulness of the combined small- and medium-angle X-ray scattering (SMAXS), which provides structural information at higher spatial resolution than commonly-used small-angle X-ray scattering, was investigated using computer simulations. For this purpose, various conformations of a protein that consists of two well-folded domains connected by a flexible linker were generated while the linker was modeled either to contain several α-helices or as random coils. The differences in the SMAXS scattering curves of these models were then evaluated. Analysis showed that the SMAXS curves allow to distinguish α-helices from random coils if at least ~20% of all the residues in the proteins contributes to the helical formation in the disordered region, suggesting that structural analysis using SMAXS will be useful not only to obtain the three-dimensional domain organizations but also to gain information on the helical propensity in the disordered regions. This would lead to more accurate interpretation of reaction kinetics that the flexible proteins are involved in.

Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated, and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.

Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using incoherent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of intermediate scattering functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.

Lysozyme amyloidosis is a hereditary severe disease, where lysozyme amyloid deposits in liver and kidney cause massive hemorrhage, resulting in the death of a patient. Amyloid fibrils are self-assembled protein filaments with core regions rich in β-sheet. It has been shown that lysozyme fibrils show structural polymorphism depending on fibrillation conditions and each polymorph shows different levels of cytotoxicity: fibrils formed at neutral pH are more cytotoxic than those formed at acidic pH. Cytotoxicity originates from interactions between fibrils and cell membranes. In particular, it is the amino acid side-chains of the fibrils that directly interact with the components of the membranes. It is thus important to characterize the mobility of side-chains of lysozyme polymorphs to gain insights into the molecular mechanism of cytotoxicity. In this study, we focused on the polymorphism of hen egg white lysozyme (HEWL), a model for studying lysozyme amyloidosis in humans and prepared D2O hydrated powder samples of two HEWL amyloid polymorphs formed at pH 6.0 (hereafter called Fib6) or 2.7 (Fib2), which are known to show higher and lower levels of cytotoxicity, respectively. We carried out elastic incoherent neutron scattering (EINS) measurements on these samples using the IN13 spectrometer at Institut Laue-Langevin in France in the temperature range from 20 K to 310 K. In the first analysis, temperature dependences of the mean square displacements (MSDs) of atomic motions in the proteins were evaluated from the EINS spectra. There were, however, no significant differences in the extracted MSD values between the two samples in the temperature range studied albeit the difference in the shape of the EINS curves. Next, analysis based on the mean square positional fluctuations (MSPFs) was conducted, which uses all the data points in the EINS curves and thus provides more detailed dynamical information than the MSD analysis. It was found that whereas the MSPF values of atomic motions are similar between the two samples, each polymorph shows a different degree of motional heterogeneity: Fib6 showed a larger fraction of atoms undergoing motions with larger amplitudes than Fib2. Furthermore, application of the Bicout -Zaccai model showed that ΔG of atomic motions tends to be lower for Fib6, suggesting that the energy barrier of atomic motions is lower for Fib6. These results suggest that the differences in molecular dynamics between lysozyme polymorphs lie in the distribution of local atomic motions and these dynamical differences would modulate the way by which the polymorphs interact with cell membranes, contributing to the differences in the level of cytotoxicity., Journées de la Diffusion Neutronique 2021

Clock proteins maintain constant enzymatic activity regardless of temperature, even though thermal fluctuation is accelerated as temperature increases. We investigated temperature influences on the dynamics of KaiC, a temperature-compensated ATPase in the cyanobacterial circadian clock system, using quasielastic neutron scattering. The frequency of picosecond to sub-nanosecond incoherent local motions in KaiC was accelerated very slightly in a temperature-dependent manner. Our mutation studies revealed that internal motions of KaiC include several contributions of opposing temperature sensitivities. To take advantage of this balancing effect, the motional frequency of local dynamics in KaiC needs to exceed ∼0.3 ps-1. Some of the mutation sites may be in a pathway through which the motional frequency in the C-terminal domain of KaiC is fed back to the active site of ATPase in its N-terminal domain. The temperature-compensating ability at the dynamics level is likely crucial for circadian clock systems, into which the clock proteins are incorporated, to achieve reaction- or even system-level temperature compensation of the oscillation frequency.

Biological membranes are generally formed by lipids and proteins. Often, the membrane properties are studied through model membranes formed by phospholipids only. They are molecules composed by a hydrophilic head group and hydrophobic tails, which can present a panoply of various motions, including small localized movements of a few atoms up to the diffusion of the whole lipid or collective motions of many of them. In the past, efforts were made to measure these motions experimentally by incoherent neutron scattering and to quantify them, but with upcoming modern neutron sources and instruments, such models can now be improved. In the present work, we expose a quantitative and exhaustive study of lipid dynamics on DMPC and DMPG membranes, using the Matryoshka model recently developed by our group. The model is confronted here to experimental data collected on two different membrane samples, at three temperatures and two instruments. Despite such complexity, the model describes reliably the data and permits to extract a series of parameters. The results compare also very well to other values found in the literature.Abstract FigureFigure 1:Graphical abstract.Highlights- The Matryoshka model brings a new general description of local lipid dynamics.- Phospholipid membranes on various conditions are compared in this novel framework.- Effects of main phase transition, membrane geometry or motion direction are probed.- Despite high number of parameters, overfitting is avoided by a global fit strategy.

In accompanying papers [Bicout et al., BBA – Biomembr. Submitted; Cissé et al., BBA – Biomembr. Submitted], a new model called Matryoshka model has been proposed to describe the geometry of atomic motions in phospholipid molecules in bilayers and multilamellar vesicles based on their quasielastic neutron scattering (QENS) spectra. Here, in order to characterize the relaxational aspects of this model, the energy widths of the QENS spectra of the samples were analyzed first in a model-free way. The spectra were decomposed into three Lorentzian functions, which are classified as slow, intermediate, and fast motions depending on their widths. The analysis provides the diffusion coefficients, residence times, and geometrical parameters for the three classes of motions. The results corroborate the parameter values such as the amplitudes and the mobile fractions of atomic motions obtained by the application of the Matryoshka model to the same samples. Since the current analysis was carried out independently of the development of the Matryoshka model, the present results enhance the validity of the model. The model will serve as a powerful tool to decipher the dynamics of lipid molecules not only in model systems, but also in more complex systems such as mixtures of different kinds of lipids or natural cell membranes.

Lipid membranes are at the basis of cell organization. Understanding their functionality, in the light of their structure and dynamics is then crucial. Using quasi-elastic neutron scattering (QENS), it is possible to access picosecond timescale dynamics. However, since years, only few models addressed the question of local lipid dynamics, whereas the increase of the neutron flux and data quality called for novel general models. Based on a timescale separation approach from Wanderlingh et al (2014), the Bicout model re-introduces the main local motions presented in the 80s by Pfeiffer et al, but writes them all in a unique framework that requires few hypotheses. Among others are described motions of the whole lipid, like its rotation, diffusion within the membrane, or in- out-of-the-plane dynamics. In addition, it makes a distinction between the head and the tail dynamics. Here, we would like to present a quantitative and complete study of local lipid dynamics on standard DMPC membranes. We probed the effects of known features such as the main phase transition, membrane geometry, or direction of motions, and proved the application of this new model., 13th European Biophysics Congress

Amyloid fibrils are self-assembled protein filaments, the deposition of which in tissues causes amyloidosis. Recently, much attention has been paid to polymorphism, where proteins form various amyloid fibrils that differ in structure and show different levels of cytotoxicity depending on fibrillation conditions. Since intramolecular motions in the fibrils are considered to play a crucial role in amyloidosis, it is important to understand the dynamical features of their polymorphs. In this study, we focused on the polymorphism of hen egg white lysozyme ( HEWL), a model for studying lysozyme amyloidosis in human and prepared D2O hydrated powder samples of two HEWL amyloid polymorphs formed at pH6 or 2, which are known to show distinct levels of cytotoxicity. We carried out the elastic incoherent neutron scattering (EINS) measurements on these samples using the IN13 spectrometer at ILL in France in the temperature range from 20 K to 310 K. The temperature dependences of the mean square displacements (MSDs) of atomic motions in the proteins were evaluated from the EINS spectra. It was found that whereas the MSD values are similar between the two fibrils at lower temperatures, the fibrils with higher toxicity show significantly larger MSDs at higher temperatures (>> 270 K). This implies that the differences in the anharmonic, diffusive local motions are related to the differences in the level of cytotoxicity. At the conference, more detailed results will be presented., MLZ Conference 2021: Neutrons for Life Sciences

Alpha-synuclein (aSyn) is an intrinsically disordered protein (IDP) that can form amyloid fibrils. Fibrils of aSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of aSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of aSyn is sensitive to solution conditions, suggesting that fibril formation of aSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because IDPs such as aSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering (QENS) and small-angle X-ray scattering (SAXS) techniques, we investigated the dynamic and structural properties of aSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The SAXS measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The QENS measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of aSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

Small-angle X-ray scattering experiments were carried out to investigate the structural changes of cardiac thin filaments induced by the cardiomyopathy-causing E244D mutation in troponin T (TnT). We examined native thin filaments (NTF) from a bovine heart, reconstituted thin filaments containing human cardiac wild-type Tn (WTF), and filaments containing the E244D mutant of Tn (DTF), in the absence and presence of Ca2+. Analysis by model calculation showed that upon Ca2+-activation, tropomyosin (Tm) and Tn in the WTF and NTF moved together in a direction to expose myosin-binding sites on actin. On the other hand, Tm and Tn of the DTF moved in the opposite directions to each other upon Ca2+-activation. These movements caused Tm to expose more myosin-binding sites on actin than the WTF, suggesting that the affinity of myosin for actin is higher for the DTF. Thus, the mutation-induced structural changes in thin filaments would increase the number of myosin molecules bound to actin compared with the WTF, resulting in the force enhancement observed for the E244D mutation.

Fluid lipid bilayers are the building blocks of biological membranes. Although there is a large amount of experimental data using inconsistent quasi-elastic neutron scattering (QENS) techniques to study membranes, very little theoretical works have been developed to study the local dynamics of membranes. The main objective of this work is to build a theoretical framework to study and describe the local dynamics of lipids and derive analytical expressions of inconsistent diffusion functions (ISF) for QENS. As results, we developed the dynamical Matryoshka model which describes the local dynamics of lipid molecules in membrane layers as a nested hierarchical convolution of three motional processes: (i) individual motions described by the vibrational motions of H-atoms; (ii) internal motions including movements of the lipid backbone, head groups and tails, and (iii) molecule movements of the lipid molecule as a whole. The analytical expressions of the ISF associated with these movements are all derived. For use in analyzing the QENS experimental data, we also derived an analytical expression for the aggregate ISF of the Matryoshka model which involves an elastic term plus three inelastic terms of well-separated time scales and whose amplitudes and rates are functions of the lipid motions. And as an illustrative application, we used the aggregated ISF to analyze the experimental QENS data on a lipid sample of multilamellar bilayers of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine). It is clear from this analysis that the dynamical Matryoshka model describes very well the experimental data and allow extracting the dynamical parameters of the studied system.

It is widely accepted that disordered regions in proteins, part of which takes predominantly α-helical conformation to a varying degree, play a critical role in biological function. Structural analysis of these flexible proteins is, however, not straightforward because existing methods to characterize the structural features of the disordered regions are not applicable to all the proteins with various sizes and physicochemical properties. In this study, to gain information on the helical propensity in the disordered regions as well as the overall three-dimensional structures of the proteins, usefulness of the combined small- and medium-angle X-ray scattering (SMAXS), which provides structural information at higher spatial resolution than commonly-used small-angle X-ray scattering, was investigated using computer simulations. For this purpose, various conformations of a protein that consists of two well-folded domains connected by a flexible linker were generated while the linker was modeled either to contain several α-helices or as random coils. The differences in the SMAXS scattering curves of these models were then evaluated. Analysis showed that the SMAXS curves allow to distinguish α-helices from random coils if at least ∼20% of all the residues in the proteins contributes to the helical formation in the disordered region, suggesting that structural analysis using SMAXS will be useful not only to obtain the three-dimensional domain organizations but also to gain information on the helical propensity in the disordered regions. This would lead to more accurate interpretation of reaction kinetics that the flexible proteins are involved in.

Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side-chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.

To study the effects of the interdomain flexibility on the encounter rate of nucleocapsid-type protein with drug molecules, where two domains (NTD) are connected by a flexible linker and each NTD has a drug binding site, two-dimensional random walk simulation was carried out as a function of the interdomain flexibility and the drug concentration. NTDs represented as circles undergo random motions constrained by the interdomain flexibility while drug molecules are represented by lattice points. It was found that as the interdomain flexibility increases, the time interval between the drug bindings to the 1st and 2nd NTDs decreases, suggesting that the 2nd drug binding is accelerated. Furthermore, this effect was more significant at lower drug concentrations. These results suggest that the interdomain linker plays a key role in the drug binding process and thus emphasize the importance of characterization of their physicochemical properties to better evaluate the efficacy of potential drugs., Graphical abstract Unlabelled Image

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D2O- and H2O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (DT), the residence time (τT), and the rotational correlation time (τR) were evaluated. The DT value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τT values were similar between S1 and F-actin. On the other hand, the τR values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the DT and τR values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins., Highlights • Hydration water dynamics of F-actin and myosin S1 was studied by neutron scattering. • Both translational and rotational motions are higher for F-actin hydration water. • Mobility of F-actin hydration water is close to that of bulk water. • High mobility of F-actin hydration water would promote the actomyosin interaction.

Troponin (Tn), consisting of three subunits, TnC, TnI, and TnT, is a protein in the thin filaments in muscle, and, together with another thin-filament protein tropomyosin (Tm), plays a major role in regulation of muscle contraction. Various mutations of Tn cause familial hypertrophic cardiomyopathy. These mutations are directly related to aberrations in this regulatory mechanism. Here we focus on the mutations E244D and K247R of TnT, which reside in the middle of the pathway of the Ca(2+)-binding signal from TnC to Tm. These mutations induce an increase in the maximum tension of cardiac muscle without changes in Ca(2+)-sensitivity. As a first step toward elucidating the molecular mechanism underlying this functional aberration, we carried out small-angle X-ray scattering experiments on the Tn core domain containing the wild type subunits and those containing the mutant TnT in the absence and presence of Ca(2+). Changes in the overall shape induced by the mutations were detected for the first time by the changes in the radius of gyration and the maximum dimension between the wild type and the mutants. Analysis of the scattering curves by model calculations shows that TnC adopts a dumbbell structure regardless of the mutations, and that the mutations change the distributions of the conformational ensembles so that the flexible N- and C-terminal regions of TnT become close to the center of the whole moelcule. This suggests, since these regions are related to the Tn-Tm interactions, that alteration of the Tn-Tm interactions induced by the mutations causes the functional aberration.

Hemoglobin, the vital O2 carrier in red blood cells, has long served as a classic example of an allosteric protein. Although high-resolution X-ray structural models are currently available for both the deoxy tense (T) and fully liganded relaxed (R) states of hemoglobin, much less is known about their dynamics, especially on the picosecond to subnanosecond time scales. Here, we investigate the picosecond dynamics of the deoxy and CO forms of human hemoglobin using quasielastic neutron scattering under near physiological conditions in order to extract the dynamics changes upon ligation. From the analysis of the global motions, we found that whereas the apparent diffusion coefficients of the deoxy form can be described by assuming translational and rotational diffusion of a rigid body, those of the CO form need to involve an additional contribution of internal large-scale motions. We also found that the local dynamics in the deoxy and CO forms are very similar in amplitude but are slightly lower in frequency...

Troponin (Tn), consisting of three subunits (TnC, TnI, and TnT), regulates cardiac muscle contraction in a Ca2+-dependent manner. Various point mutations of human cardiac Tn are known to cause familial hypertrophic cardiomyopathy due to aberration of the regulatory function. In this study, we investigated the effects of one of these mutations, K247R of TnT, on the picosecond dynamics of the Tn core domain (Tn-CD), consisting of TnC, TnI and TnT2 (183–288 residues of TnT), by carrying out the quasielastic neutron scattering measurements on the reconstituted Tn-CD containing either the wild-type TnT2 (wtTn-CD) or the mutant TnT2 (K247R-Tn-CD) in the absence and presence of Ca2+. It was found that Ca2+-binding to the wtTn-CD decreases the residence time of atomic motions in the Tn-CD with slight changes in amplitudes, suggesting that the regulatory function mainly requires modulation of frequency of atomic motions. On the other hand, the K247R-Tn-CD shows different dynamic behavior from that of the wtTn-CD both in the absence and presence of Ca2+. In particular, the K247R-Tn-CD exhibits a larger amplitude than the wtTn-CD in the presence of Ca2+, suggesting that the mutant can explore larger conformational space than the wild-type. This increased flexibility should be relevant to the functional aberration of this mutant.