Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

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 (D_T), the residence time (τ_T), and the rotational correlation time (τ_R) were evaluated. The D_T value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τ_T values were similar between S1 and F-actin. On the other hand, the τ_R 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 D_T and τ_R 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.     

Picosecond dynamical changes on denaturation of yeast phosphoglycerate kinase revealed by quasielastic neutron scattering

Quasielastic neutron scattering experiments performed on yeast phosphoglycerate kinase in the native form and denatured in 1.5 M guanidinium chloride reveal a change in the fast (picosecond time scale) diffusive internal dynamics of the protein. The momentum and energy transfer dependences of the scattering for both states are fitted by an analytical model in which, on the experimentally accessible picosecond time scale and angstrom length scale, the dynamics of a fraction of the nonexchangeable hydrogens in the protein is described as a superposition of vibrations with uniform diffusion in a sphere, the rest of the hydrogens undergoing only vibrational motion. The fraction diffusing changes, from ≈60% in the native protein to ≈82% in the denatured protein. The radius of the sphere also changes slightly, from ≈1.8 Å in the native protein to ≈2.2 Å in the denatured protein. Possible implications of these results for the general protein folding problem are discussed. Proteins 28:380–387, 1997 © 1997 Wiley-Liss, Inc.     

Time-resolved quasielastic neutron scattering studies of native photosystems

The internal molecular dynamics of proteins plays an important role in a number of functional processes in native photosystems. Prominent examples include the photocycle of bacteriorhodopsin and electron transfer in the reaction center of plant photosystem II. In this regard, the recently developed technique of time-resolved quasielastic neutron scattering with laser excitation opens up new perspectives for the study of protein/membrane dynamics in specific functional states of even complex systems. The first direct observation of a functionally modulated protein dynamics has just recently been reported for the model system bacteriorhodopsin (Pieper et al., Phys. Rev. Lett. 100, 2008, 228103.), where a transient softening of the protein was observed on a timescale of ∼ 1 ms along with the large-scale structural change in the M-intermediate of bacteriorhodopsin. In contrast, photosystem II membrane fragments with inhibited electron transfer show a suppression of protein dynamics ∼160 μs after the actinic laser flash (Pieper and Renger, Biochemistry 48, 2009, 6111). This effect may reflect aggregation-like conformational changes capable of dissipation of excess excitation energy to prevent photodamage in the absence of Q_A→Q_B electron transfer. These findings indicate that proteins exhibit a remarkable flexibility to accommodate different functional processes. This contribution will discuss methodical aspects, challenges, and recent applications of laser-excited, time-resolved quasielastic neutron scattering.     

Self-similar dynamics of proteins under hydrostatic pressure—Computer simulations and experiments

Different experimental techniques, such as kinetic studies of ligand binding and fluorescence correlation spectroscopy, have revealed that the diffusive, internal dynamics of proteins exhibits autosimilarity on the time scale from microseconds to hours. Computer simulations have demonstrated that this type of dynamics is already established on the much shorter nanosecond time scale, which is also covered by quasielastic neutron scattering experiments. The autosimilarity of protein dynamics is reflected in long-time memory effects in the underlying diffusion processes, which lead to a non-exponential decay of the observed time correlation functions. Fractional Brownian dynamics is an empirical model which is able to capture the essential aspects of internal protein dynamics. Here we give a brief introduction into the theory and show how the model can be used to interpret neutron scattering experiments and molecular dynamics simulation of proteins in solution under hydrostatic pressure.     

Protein dynamics in solution and powder measured by incoherent elastic neutron scattering: the influence of Q-range and energy resolution

Incoherent elastic neutron scattering (IENS) has been widely used to measure intramolecular atomic mean square displacements (MSDs) of proteins in powder and in solution. The instrumental energy resolution and the wave vector transfer (Q-range) determine, respectively, the time and length scales of observable motions. In order to investigate contributions of diffusive motions to MSDs measured by this method, we calculated the elastic intensity for several simple scattering functions. We showed that continuous translational diffusion contributes to MSDs in a Q-range where the energy width of the scattering function is of the order of the instrumental energy resolution. We discuss the choice of instruments adapted to focus on intramolecular motions in the presence of solvent or global macromolecular diffusion. The concepts developed are applied to interpret experimental data from H₂O- and D₂O-hydrated proteins. Finally, analogies between the Gaussian approximation in IENS and the Guinier approximation in small-angle scattering are discussed.     

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations.     

Picosecond time-resolved X-ray crystallography: probing protein function in real time

A detailed mechanistic understanding of how a protein functions requires knowledge not only of its static structure, but also how its conformation evolves as it executes its function. The recent development of picosecond time-resolved X-ray crystallography has allowed us to visualize in real time and with atomic detail the conformational evolution of a protein. Here, we report the photolysis-induced structural evolution of wild-type and L29F myoglobin over times ranging from 100 ps to 3 micros. The sub-ns structural rearrangements that accompany ligand dissociation in wild-type and the mutant form differ dramatically, and lead to vastly different ligand migration dynamics. The correlated protein displacements provide a structural explanation for the kinetic differences. Our observation of functionally important protein motion on the sub-ns time scale was made possible by the 150-ps time resolution of the measurement, and demonstrates that picosecond dynamics are relevant to protein function. To visualize subtle structural changes without modeling, we developed a novel method for rendering time-resolved electron density that depicts motion as a color gradient across the atom or group of atoms that move. A sequence of these time-resolved images have been stitched together into a movie, which allows one to literally "watch" the protein as it executes its function.     

Coupling of laser excitation and inelastic neutron scattering: attempt to probe the dynamics of light-induced C-phycocyanin dynamics

Excitation energy transfer (EET) in light-harvesting antennae is a highly efficient key event in photosynthesis, where light-induced dynamics of the antenna pigment-protein complexes may play a functional role. So far, however, the relationship between EET and protein dynamics remains unknown. C-phycocyanin (C-PC) is the main pigment/protein complex present in the cyanobacterial antenna, called "phycobilisome". The aim of the present study was to investigate light-induced C-PC internal thermal motions (ps timescale) measured by inelastic neutron scattering. To synchronize the beginning of the laser flash (6 ns duration) with that of the neutron test pulse ( approximately 87 mus duration), we developed a novel type of "time-resolved" experimental setup on MIBEMOL time-of-flight neutron spectrometer (LLB, France). Data acquisition has been modified to get quasi-simultaneously "light" and "dark" measurements (with and without laser, respectively) and eliminate many spurious effects that could occur on the sample during the experiment. The study was carried out on concentrated C-PC ( approximately 135 g/L protein in D(2)O phosphate buffer), contained in an aluminium/sapphire sample holder (almost "transparent" for neutrons) and homogeneously illuminated inside an "integrating sphere". We observed very similar incoherent dynamical structure factors of C-PC with or without light. The vibrational density of states showed two very slightly increased vibrational modes with light, at approximately 30 and approximately 50 meV ( approximately 240 and approximately 400 cm(-1), respectively). These effects have to be verified by further experiments before probing any temporal evolution, by introducing a time delay between the laser flash and the neutron test pulse.     

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Deuterated iodoacetanilide: A covalent protein label for neutron scattering studies

A simple preparation of a deuterated alkylating reagent is described. Its reaction with the β⁹³-SH groups of hemoglobin was used to illustrate the preparation of a protein derivative which contains 5 covalently attached non-exchangeble deuterium atoms at a unique locus on each β chain.     

The effect of hydration on protein flexibility in photosystem II of green plants studied by quasielastic neutron scattering

The effect of hydration on protein dynamics in photosystem II (PS II) membrane fragments from spinach has been investigated by using the method of quasielastic neutron scattering (QENS) at room temperature. The QENS data obtained indicate that the protein dynamics is strongly dependent on the extent of hydration. In particular, the hydration-induced activation of localized diffusive protein motions and Q_A⁻ reoxidation by Q_B in PS II appear to be correlated in their onset at a hydration value of about 45% relative humidity (r.h.). These findings underline the crucial functional relevance of localized diffusive protein motions on the picosecond-timescale for the reactions of light-induced photosynthetic water splitting under formation of plastoquinol and molecular oxygen in PS II of green plants. Advanced neutron scattering and complementary techniques to study biological systems. Contributions from the meetings, “Neutrons in Biology”, STFC Rutherford Appleton Laboratory, Didcot, UK, 11–13 July and “Proteins At Work 2007”, Perugia, Italy, 28–30 May 2007.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

Structural themes and variations in protein kinase A as seen by small-angle scattering and neutron contrast variation

Using small-angle solution scattering and neutron contrast variation, we have studied the structure of the multi-subunit protein kinase A. We have gained insights into how nature can take a set of common structural domains (or themes) and modulate their interactions via sequence variations and second messenger mediated signaling to affect enzyme activity and receptor binding important for targeting this multi-function enzyme to specific sub-cellular locations. These studies demonstrate the power of neutron contrast variation to expand our knowledge of the dynamic supra-molecular structures that carry out biological function.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.     

Influence of pressure on the low‐frequency vibrational modes of lysozyme and water: A complementary inelastic neutron scattering and molecular dynamics simulation study

We performed complementary inelastic neutron scattering (INS) experiments and molecular dynamics (MD) simulations to study the influence of pressure on the low‐frequency vibrational modes of lysozyme in aqueous solution in the 1 atm–6 kbar range. Increasing pressure induces a high‐frequency shift of the low‐frequency part (<10 meV = 80 cm^?1) of the vibrational density of states (VDOS), g(ω), of both lysozyme and water that reveals a stiffening of the interactions ascribed to the reduction of the protein and water volumes. Accordingly, high pressures increase the curvature of the free energy profiles of the protein quasiharmonic vibrational modes. Furthermore, the nonlinear influence of pressure on the g(ω) of lysozyme indicates a change of protein dynamics that reflects the nonlinear pressure dependence of the protein compressibility. An analogous dynamical change is observed for water and stems from the distortion of its tetrahedral structure under pressure. Moreover, our study reveals that the structural, dynamical, and vibrational properties of the hydration water of lysozyme are less sensitive to pressure than those of bulk water, thereby evidencing the strong influence of the protein surface on hydration water. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

The vibrational spectrum of TpRhH2(H2): a computational and inelastic neutron scattering study

We report extensive density functional theory studies of the structures and vibrational frequencies of Tp^3,5-MeRhH₂(H₂) in its ground and various transition states as well as the first direct comparison of observed and calculated inelastic neutron scattering (INS) vibrational spectra on this type of compound. Geometry optimizations produced canted η²-dihydrogen dihydride local minima of C₁ symmetry; with HH distances for the C₁ minimum energy structure of 0.842 and 0.898 Å and barriers to rotation of 0.34 and 0.50 kcal mol⁻¹, respectively for B3LYP/BS1 and BP86/BS1 calculations of Tp^3,5-MeRhH₂(H₂). The latter results from one transition state rotated approximately 60° away (a second lower energy transition state which is a few hundreds of a kcal mol⁻¹ above the C₁ MIN is rotated approximately 30° away). With these calculated d(HH) values for the C₁ MIN the previously reported experimental data on the rotation of the dihydrogen ligand yields an experimental barrier to rotation of 1 kcal mol⁻¹ and places the torsional transition at 200 cm⁻¹ in the INS spectrum. Optimization of the Rh structure, that is analogous to the related Ir(V) C_s minimum found for TpIrH₄, generates a high-energy (>4 cal mol⁻¹) C_s transition state TpRh^IIIH₄ structure with an η³-H₃ ⁻ ligand. This transition state (C_s TSE) exchanges the hydrogen in the mirror plane between two chiral C₁ MIN structures. Comparisons between observed and computed INS spectra suggests that the experimental INS spectrum be viewed as resulting from a quantum-averaged ground state encompassing at least two of the low energy structures found in our calculations.     

C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions

We present a study of C-phycocyanin hydration water dynamics in the presence of trehalose by incoherent elastic neutron scattering. By combining data from two backscattering spectrometers with a 10-fold difference in energy resolution we extract a scattering law S(Q,omega) from the Q-dependence of the elastic intensities without sampling the quasielastic range. The hydration water is described by two dynamically different populations--one diffusing inside a sphere and the other diffusing quasifreely--with a population ratio that depends on temperature. The scattering law derived describes the experimental data from both instruments excellently over a large temperature range (235-320 K). The effective diffusion coefficient extracted is reduced by a factor of 10-15 with respect to bulk water at corresponding temperatures. Our approach demonstrates the benefits and the efficiency of using different energy resolutions in incoherent elastic neutron scattering over a large angular range for the study of biological macromolecules and hydration water.     

Large scale motions in a biosensor protein glucose oxidase: a combined approach by QENS, normal mode analysis, and molecular dynamics studies

The characteristics of the glucose oxidase were studied using a combination of experimental and theoretical techniques. Quasi elastic neutron scattering experiments were used to obtain the vibrational frequencies of the protein. These were compared to theoretical results obtained by normal mode analysis. Results indicate a good match between the experimental and theoretical values. Molecular dynamic simulation with covariant analysis was used to study the structure and dynamics of glucose oxidase. Various parameters like the radius of gyration, root mean square fluctuations, solvent accessibility were studied for evaluating the structural stability of the protein. The frequency of vibration calculated from the three methods is used to derive the large scale motions. Theses studies were used to predict the suitable lysine residues for linkage with carbon nanotubes.     

Structure from NMR and molecular dynamics: Distance restraining inhibits motion in the essential subspace

Summary We address the question how well proteins can be modelled on the basis of NMR data, when these data are incorporated into the protein model using distance restraints in a molecular dynamics simulation. We found, using HPr as a model protein, that distance restraining freezes the essential motion of proteins, as defined by Amadei et al. [Amadei, A., Linssen, A.B.M. and Berendsen, H.J.C. (1993) Protein Struct. Funct. Genet., 17, 412–425]. We discuss how modelling protocols can be improved in order to solve this problem.     

The MS2 coat protein shell is likely assembled under tension: a novel role for the MS2 bacteriophage A protein as revealed by small-angle neutron scattering

Recombinant forms of the bacteriophage MS2 and its RNA-free (empty) MS2 capsid were analyzed in solution to determine if RNA content and/or the A (or maturation) protein play a role in the global arrangement of the virus protein shell. Analysis of the (coat) protein shell of recombinant versions of MS2 that lack the A protein revealed dramatic differences compared to wild-type MS2 in solution. Specifically, A protein-deficient virus particles form a protein shell of between 31(+/-1) A and 37(+/-1) A. This is considerably thicker than the protein shell formed by either the wild-type MS2 or the RNA-free MS2 capsid, whose protein shells have a thickness of 21(+/-1) A and 25(+/-1) A, respectively. Since the A protein is known to separate from the intact MS2 protein shell after infection, the thin shell form of MS2 represents the pre-infection state, while the post-infection state is thick. Interestingly, these A protein-dependent differences in the virus protein shell are not seen using crystallography, as the crystallization process seems to artificially compact the wild-type MS2 virion. Furthermore, when the A protein is absent from the virus shell (post-infection), the process of crystallization exerts sufficient force to convert the protein shell from the post-infection (thick) state to the pre-infection (thin) conformation. In summary, the data are consistent with the idea that RNA content or amount does not affect the structure of the MS2 virus shell. Rather, the A protein influences the global arrangement of the virus coat dramatically, possibly by mediating the storage of energy or tension within the protein shell during virus assembly. This tension may later be used to eject the MS2 genomic RNA and A protein fragments into the host during infection.