Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

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High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes.

The influence of high hydrostatic pressure on the internal sub-nanosecond dynamics of highly concentrated lysozyme in aqueous solutions was studied by elastic incoherent neutron scattering (EINS) up to pressures of 4 kbar. We have found, with increasing pressure, a reduction in the dynamics of H atoms of folded lysozyme, suggesting a loss in protein mobility that follows a change in the local energy landscape upon the increase in packing density. Moreover, the amplitude of the protein fluctuations depends drastically on the protein concentration, and protein structural and interaction parameters as well as the dynamical properties are affected by pressure in a nonlinear way. A significant reduction of the mean squared displacement of H atoms occurs already at rather low pressures of a few hundred bars for lysozyme in bulk water solution. This trend is lifted at ∼2 kbar, which is probably due to a solvent-mediated effect. Conversely, for high protein concentrations (e.g., 160 mg mL–1), that is, under stron...

Influence of pressure and crowding on the sub-nanosecond dynamics of globular proteins.

We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein.

Pressure-induced molten globule state of human acetylcholinesterase: structural and dynamical changes monitored by neutron scattering

The effects of high hydrostatic pressure on the structure and dynamics of model membrane systems were investigated using neutron scattering. Diffraction experiments show shifts of the pre- and main-phase transitions of multilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to higher temperatures with increased pressure which are close to results observed previously by other techniques, namely (10.4 ± 1.0) K kbar(-1) and (20.0 ± 0.5) K kbar(-1) for the two transitions. Backscattering spectroscopy reveals that the mean square displacements in the liquid phase are about 10% smaller at 300 bar and about 20% smaller at 600 bar compared to atmospheric pressure, whereas in the gel phase below the main phase transition the mean square displacements show a smaller difference in the dynamics of the three pressure values within the studied pressure range.

High hydrostatic pressure effects investigated by neutron scattering on lipid multilamellar vesicles

We studied the effects of temperature and hydrostatic pressure on the dynamical properties and folding stability of highly concentrated lysozyme solutions in the absence and presence of the osmolytes trimethylamine-N-oxide (TMAO) and urea. Elastic incoherent neutron scattering (EINS) was applied to determine the mean-squared displacement (MSD) of the protein's hydrogen atoms to yield insights into the effects of these cosolvents on the averaged sub-nanosecond dynamics in the pressure range from ambient up to 4000 bar. To evaluate the additional effect of self-crowding, two protein concentrations (80 and 160 mg mL-1) were used. We observed a distinct effect of TMAO on the internal hydrogen dynamics, namely a reduced mobility. Urea, on the other hand, revealed no marked effect and consequently, no counteracting effect in an urea-TMAO mixture was observed. Different from the less concentrated protein solution, no significant effect of pressure on the MSD was observed for 160 mg mL-1 lysozyme. The EINS experiments were complemented by Fourier-transform infrared (FTIR) spectroscopy measurements, which led to additional insights into the folding stability of lysozyme under the various environmental conditions. We observed a stabilization of the protein in the presence of the compatible osmolyte TMAO and a destabilization in the presence of urea against temperature and pressure for both protein concentrations. Additionally, we noticed a slight destabilizing effect upon self-crowding at very high protein concentration (160 mg mL-1), which is attributable to transient destabilizing intermolecular interactions. Furthermore, a pressure-temperature diagram could be obtained for lysozyme at these high protein concentrations that mimics densely packed intracellular conditions.

Influence of cosolvents, self-crowding, temperature and pressure on the sub-nanosecond dynamics and folding stability of lysozyme

The design of new high pressure equipment for structural and dynamical studies on samples in solution is described. We present two sample cells for applying pressures up to 150 and 700 MPa (i.e. 1.5 and 7 kbar), respectively. These cells are mounted on special sticks and inserted into the calorimeter of a cryostat to regulate the temperature. Different parts of the equipment – the pressure controller, the sticks and the cells – are described. In addition, radiography tests which were performed with neutrons in situ at the Institut Laue Langevin to verify the tightness of the cells and the hydrostatic transmission of the pressure to the sample are presented. First results on model lipids are in agreement with former results by R. Winter et al. [Towards an understanding of the temperature/pressure configurational and free-energy landscape of biomolecules, J. Non-Equilib. Thermodyn. 32 (2007), pp. 41–97].

High hydrostatic pressure equipment for neutron scattering studies of samples in solutions

A new source of ultracold neutrons (UCNs), developed at the Institut Laue-Langevin (ILL) and named SuperSUN, is currently being commissioned. Its operational principle is the conversion of cold neutrons, delivered by ILL’s existing beam H523, to UCNs in a vessel filled with superfluid helium-4, wherein the neutron’s energy and momentum are transferred by inelastic scattering to phonons in the superfluid. The inverse Boltzmann-suppressed process is negligible at temperatures below 0.6 K, enabling long storage times and high in-situ UCN densities as demonstrated at the ILL for two prototype sources. These two prototypes are installed at secondary beams behind crystal monochromators, whereas a primary beam with a white cold spectrum illuminates the SuperSUN conversion volume. This provides not only higher intensity around the wavelength 0.89 nm where the dominant single-phonon process for UCN production takes place, but also a contribution to UCN production by multi-phonon processes. In the first phase of the project, material walls will trap the UCNs, while in the second phase an octupole magnet will generate a 2.1 T magnetic field at the edge of the conversion volume. For low-field-seeking UCNs, this field increases the trapping potential and reduces wall losses so that the accumulated UCNs are spin-polarized as a result. SuperSUN aims to deliver the highest possible UCN densities to external storage experiments, the first of which will be the PanEDM experiment measuring the neutron’s permanent electric dipole moment.

Concept and strategy of SuperSUN: A new ultracold neutron converter

Blue series top-loading furnaces are regularly used at neutron facilities to carry out experiments at up to 1700◦C. The sample temperature is controlled using the indirect resistance heating method which consists in placing the sample inside a radiating cylindrical heater made from vanadium or niobium into which a current of up to 180 A circulates. To prevent the oxydation and deterioration of the heated elements, the sample, the heater and the heat shields are in a secondary vacuum which limits very much the cooling rate. We present an automated fast-cooling technique which reduces the cool-down time by a factor of 4 to 5 without impacting the lifetime of the heated elements. This method is now routinely used at the ILL and ISIS facilities and reduces very much beam time losses.

Nadir Belkhier

Papers

High hydrostatic pressure equipment for neutron scattering studies of samples in solutions

Concept and strategy of SuperSUN: A new ultracold neutron converter

A fast-cooling mode for blue series furnaces