Picosecond molecular motions in bacteriorhodopsin from neutron scattering (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 1996
Quasielastic incoherent neutron scattering from hydrogen atoms, which are distributed nearly homogeneously in biological molecules, allows the investigation of diffusive motions occurring on the picoto nanosecond time scale. A quasielastic incoherent neutron scattering study was performed on the integral membrane protein bacteriorhodopsin (BR), which is a light-driven proton pump in Halobacterium salinarium. BR is embedded in lipids, forming patches in the cell membrane of the organism, which are the so called purple membranes (PMs). Measurements were carried out at room temperature on oriented PM-stacks hydrated at two different levels (low hydration, h = 0.03 g of D20 per g of PM; high hydration, h = 0.28 g of D20 per g of PM) using time-of-flight spectrometers. From the measured spectra, different diffusive components were identified and analyzed with respect to the influence of hydration. This study supports the idea that a decrease in hydration results in an appreciable decrease in internal molecular flexibility of the protein structure. Because it is known from studies on the function of BR that the pump activity is reduced if the hydration level of the protein is insufficient, we conclude that the observed diffusive motions are essential for the function of this protein. A detailed analysis and classification of the different kinds of diffusive motions, predominantly occurring in PMs under physiological conditions, is presented.
Biophysical Journal, 1998
Bacteriorhodopsin (BR) is a transmembrane protein in the purple membrane (PM) of Halobacterium salinarum. Its function as a light-driven proton pump is associated with a cycle of photointermediates which is strongly hydrationdependent. Using energy-resolved neutron scattering, we analyzed the thermal motions (in the nanosecond-to-picosecond time range) in PM at different hydration levels. Two main populations of motions were found that responded differently to water binding. Striking correlations appeared between these "fast" motions and the "slower" kinetic constants (in the millisecond time range) of relaxations and conformational changes occurring during the photocycle.
1995
The incoherent quasi-elastic neutron scattering (IQNS) method is a useful technique to study biomolecular dynamics. The versatility of the method makes possible motional studies of biomolecules in different forms: powder, crystal, and solution; and at different temperatures. Thus, it allows for the investigation of biomolecular dynamics over a wide-range of physical conditions. We have used the IQNS method to study the motions of side chains in trypsin and myoglobin at various D_2O hydration levels. The scattering spectra S(Q, omega) were measured in constant-Q mode. The protein in powder form exhibits vibrational high-frequency motions, while the protein in solution and in crystals are characterized by diffusive jumps, and high-frequency vibrations. At temperatures below 200K, the S(Q, omega) for these proteins in solution is similar to an harmonic solid. As temperature increases, a transition is seen at 200K, above which the protein becomes more liquid -like with rapid transitions between conformational substates. The diffusion constant D for the side chains is on the order of 10^{-6} cm ^2/sec.
Internal motions in proteins: A combined neutron scattering and molecular modelling approach
Pramana, 2004
It is well-known that water plays a major role in the stability and catalytic function of proteins. Both the effect of hydration water on the dynamics of proteins and that of proteins on the dynamics of water have been studied using inelastic neutron scattering. Inelastic neutron scattering is the most direct probe of diffusive protein dynamics on the picosecond-nanosecond time-scale. We present here results relative to a photosynthetic globular protein, the C-phycocyanin, that can be obtained in protonated and deuterated forms. Diffusive motions have been studied using the protonated C-phycocyanin, protein. Molecular dynamics simulation and analytical theory have been combined to analyse the data and get a detailed description of diffusive motions for protein. The simulation-derived dynamic structure factors are in good agreement with experiment. The dynamical parameters are shown to present a smooth variation with distance from the core of the protein.
Dynamics of different functional parts of bacteriorhodopsin: H-2H labeling and neutron scattering
Proceedings of the National Academy of Sciences, 1998
We show that dynamics of specific amino acids within a protein can be characterized by neutron spectroscopy and hydrogen-deuterium labeling, and we present data on the motions of a selected set of groups within bacteriorhodopsin (BR), the retinal-based proton pump in the purple membrane of halophilic Archaea. Elastic incoherent neutron scattering experiments allow the definition of motions in the nano-to picosecond time scale and have revealed a dynamical transition from a harmonic to a softer, anharmonic atomic f luctuation regime in the global behavior of proteins. Biological activity in proteins is correlated with this transition, suggesting that f lexibility is required for function. Elastic incoherent neutron scattering is dominated by H atom scattering, and to study the dynamics of a selected part of BR, fully deuterated purple membrane with BR containing Hretinal, H-tryptophan, and H-methionine was prepared biosynthetically in Halobacterium salinarum. These amino acids cluster in the functional center of the protein. In contrast to the protein globally, the thermal motions of the labeled atoms were found to be shielded from solvent melting effects at 260 K. Above this temperature, the labeled groups appear as more rigid than the rest of the protein, with a significantly smaller mean square amplitude of motion. These experimental results quantify the dynamical heterogeneity of BR (which meets the functional requirements of global f lexibility), on the one hand, to allow large conformational changes in the molecule and of a more rigid region in the protein, on the other, to control stereo-specific selection of retinal conformations.
Hydration Effect on Low-Frequency Protein Dynamics Observed in Simulated Neutron Scattering Spectra
Biophysical Journal, 2008
Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (,;20 meV) is developed in the near future.
Dynamics of Protein and its Hydration Water: Neutron Scattering Studies on Fully Deuterated GFP
Biophysical Journal, 2012
We present a detailed analysis of the picosecond-to-nanosecond motions of green fluorescent protein (GFP) and its hydration water using neutron scattering spectroscopy and hydrogen/deuterium contrast. The analysis reveals that hydration water suppresses protein motions at lower temperatures (<~200 K), and facilitates protein dynamics at high temperatures. Experimental data demonstrate that the hydration water is harmonic at temperatures <~180-190 K and is not affected by the proteins' methyl group rotations. The dynamics of the hydration water exhibits changes at~180-190 K that we ascribe to the glass transition in the hydrated protein. Our results confirm significant differences in the dynamics of protein and its hydration water at high temperatures: on the picosecond-to-nanosecond timescale, the hydration water exhibits diffusive dynamics, while the protein motions are localized to <~3 Å. The diffusion of the GFP hydration water is similar to the behavior of hydration water previously observed for other proteins. Comparison with other globular proteins (e.g., lysozyme) reveals that on the timescale of 1 ns and at equivalent hydration level, GFP dynamics (mean-square displacements and quasielastic intensity) are of much smaller amplitude. Moreover, the suppression of the protein dynamics by the hydration water at low temperatures appears to be stronger in GFP than in other globular proteins. We ascribe this observation to the barrellike structure of GFP.
Spectroscopy, 2010
This review article describes our neutron scattering experiments made in the past four years for the understanding of the single-particle (hydrogen atom) dynamics of a protein and its hydration water and the strong coupling between them. We found that the key to this strong coupling is the existence of a fragile-to-strong dynamic crossover (FSC) phenomenon occurring at around T L = 225 ± 5 K in the hydration water. On lowering of the temperature toward FSC, the structure of hydration water makes a transition from predominantly the high density form (HDL), a more fluid state, to predominantly the low density form (LDL), a less fluid state, derived from the existence of a liquid-liquid critical point at an elevated pressure. We show experimentally that this sudden switch in the mobility of hydration water on Lysozyme, B-DNA and RNA triggers the dynamic transition, at a temperature T D = 220 K, for these biopolymers. In the glassy state, below T D , the biopolymers lose their vital conformational flexibility resulting in a substantial diminishing of their biological functions. We also performed molecular dynamics (MD) simulations on a realistic model of hydrated lysozyme powder, which confirms the existence of the FSC and the hydration level dependence of the FSC temperature. Furthermore, we show a striking feature in the short time relaxation (β-relaxation) of protein dynamics, which is the logarithmic decay spanning 3 decades (from ps to ns). The long time α-relaxation shows instead a diffusive behavior, which supports the liquid-like motions of protein constituents. We then discuss our recent high-resolution X-ray inelastic scattering studies of globular proteins, Lysozyme and Bovine Serum Albumin. We were able to measure the dispersion relations of collective, intra-protein phonon-like excitations in these proteins for the first time. We found that the phonon energies show a marked softening and at the same time their population increases substantially in a certain wave vector range when temperature crosses over the T D . Thus the increase of biological activities above T D has positive correlation with activation of slower and large amplitude collective motions of a protein.
Molecular motions and hydration of purple membranes and disk membranes studied by neutron scattering
European Biophysics Journal, 1998
Fast stochastic equilibrium fluctuations (time scale: 10 -10 -10 -13 seconds) in purple membranes (PM) and in disk membranes (DM) have been measured with quasielastic incoherent neutron scattering. The comparison of predominantly stochastic motions occurring in purple membranes and in disk membranes revealed qualitatively similar dynamical behaviour. Models of internal motions within restricted volumes have been shown to be useful to fit the spectra from both samples. From fits using these models we found "amplitudes" 15 to 20% larger for motions in DM samples compared to PM samples. This indicates a higher internal flexibility of the DM. Because the dynamical behaviour is very sensitive to the hydration of the protein-lipid complex, we also performed neutron diffraction experiments to determine lamellar spacings as a measure of level of hydration and as a function of temperature. From these studies the interaction of solvent molecules with the surface of the protein-lipid complex appears to be qualitatively similar for both types of membranes.