Valérie Réat

Enzyme dynamics and activity: time-scale dependence of dynamical transitions in glutamate dehydrogenase solution.

We have examined the temperature dependence of motions in a cryosolution of the enzyme glutamate dehydrogenase (GDH) and compared these with activity. Dynamic neutron scattering was performed with two instruments of different energy resolution, permitting the separate determination of the average dynamical mean square displacements on the sub-approximately 100 ps and sub-approximately 5 ns time scales. The results demonstrate a marked dependence on the time scale of the temperature profile of the mean square displacement. The lowest temperature at which anharmonic motion is observed is heavily dependent on the time window of the instrument used to observe the dynamics. Several dynamical transitions (inflexions of the mean squared displacement) are observed in the slower dynamics. Comparison with the temperature profile of the activity of the enzyme in the same solvent reveals dynamical transitions that have no effect on GDH function.

Order Parameters of a Transmembrane Helix in a Fluid Bilayer: Case Study of a WALP Peptide

A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including (13)C and (15)N chemical shift anisotropies and (13)C-(15)N dipolar couplings, were determined from two different triple-isotope-labeled WALP23 peptides ((2)H, (13)C, and (15)N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 degrees was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 degrees previously determined from (2)H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error.

High resolution 2D 1H–13C correlation of cholesterol in model membrane

High resolution 2D NMR MAS spectra of liposomes, in particular 1H-13C chemical shifts correlations have been obtained on fluid lipid bilayers made of pure phospholipids for several years. We have investigated herein the possibility to obtain high resolution 2D MAS spectra of cholesterol embedded in membranes, i.e. on a rigid molecule whose dynamics is characterized mainly by axial diffusion without internal segmental mobility. The efficiency of various pulse sequences for heteronuclear HETCOR has been compared in terms of resolution, sensitivity and selectivity, using either cross polarization or INEPT for coherence transfer, and with or without MREV-8 homonuclear decoupling during t1. At moderately high spinning speed (9 kHz), a similar resolution is obtained in all cases (0.2 ppm for 1H(3,4), 0.15 ppm for 13C(3,4) cholesterol resonances), while sensitivity increases in the order: INEPT < CP(x4) < CP + MREV. At reduced spinning speed (5 kHz), the homonuclear dipolar coupling between the two geminal protons attached to C(4) gives rise to spinning sidebands from which one can estimate a H-H dipolar coupling of 10 kHz which is in good agreement with the known dynamics of cholesterol in membranes.

Hydrogen bonding of cholesterol in the lipidic cubic phase.

The addition of cholesterol to the monoolein-based lipidic cubic phase (LCP) has been instrumental in obtaining high-resolution crystal structures of several G protein-coupled receptors. Here, we report the use of high-resolution magic angle spinning NMR spectroscopy to record and assign the isotropic (13)C chemical shifts of cholesterol in lipidic lamellar and cubic phases at different hydration levels with monoolein and chain-deuterated DMPC as host lipids. The hydrogen-bonding patterns of cholesterol in these phases were determined from the NMR data by quantum chemical calculations. The results are consistent with the normal orientation of cholesterol in lipid bilayers and with the cholesterol hydroxyl group located at the hydrophobic/hydrophilic interface. The (13)C chemical shifts of cholesterol are mostly affected by the host lipid identity with little or no dependency on the hydration (20% vs 40%) or the phase identity (lamellar vs LCP). In chain-deuterated DMPC bilayers, the hydroxyl group of cholesterol forms most of its hydrogen bonds with water, while in monoolein bilayers it predominately interacts with monoolein. Such differences in the hydrogen-bonding network of cholesterol may have implications for the design of experiments in monoolein-based LCP.

Proton channel hydration and dynamics of a bacteriorhodopsin triple mutant with an M-state-like conformation

AbstractThe hydration and dynamics of purple membranes (PM) containing the bacteriorhodopsin (BR) triple mutant D96G/F171C/F219L were investigated by neutron diffraction coupled with H2O/D2O exchange and by energy-resolved neutron scattering. The mutant, which is active in proton transport (Tittor et al. in J. Mol. Biol. 319:555–565, 2002), has an ‘open’ ground-state structure similar to that of the M intermediate in the photocycle of the wild type (wt) (Subramaniam and Henderson in Nature 406:653–657, 2000). The experiments demonstrated an increased proton channel hydration in the mutant PM compared with wt PM, in both high (86%) and low (57%) relative humidity. We suggest that this is due to the smaller side chains of the mutant residues liberating space for more water molecules in the proton channel, which would then be able to participate in the proton translocation network. PM thermal dynamics has been shown to be very sensitive to membrane hydration (Lehnert et al. in Biophys. J. 75:1945–1952, 1998). The global dynamical behaviour of the mutant PM on the 100-ps time scale, as a function of relative humidity, was found to be identical to that of the wt, showing that the ‘open’ BR structure and additional water molecules in the proton channel do not provide a softer environment enabling increased flexibility.

Cryosolvents useful for protein and enzyme studies below −100°C

Abstract For the study of protein structure, dynamics, and function, at very low temperatures it is desirable to use cryosolvents that resist phase separation and crystallisation. We have examined these properties in a variety of cryosolvents. Using visual and X-ray diffraction criteria, methanol:ethanediol (70%:10%), methanol:glycerol (70%:10%), acetone:methoxyethanol:ethanediol (35%:35%:10%), dimethylformamide:ethanediol (70%:10%), dimethylformamide (80%), methoxyethanol (80%), and methoxyethanol:ethanediol (70%:10%) were all found to be free of phase-changes down to at least −160°C. The least viscous of these, methanol:ethanediol (70%:10%), was miscible down to −125°C and showed no exo or endothermic transitions when examined using DSC. It is therefore potentially particularly suitable for very low temperature cryoenzymology.