Water-Mediated Protein-Protein Interactions at High Pressures are Controlled by a Deep-Sea Osmolyte.

The influence of natural cosolvent mixtures on the pressure-dependent structure and protein-protein interaction potential of dense protein solutions is studied and analyzed using small-angle X-ray scattering in combination with a liquid-state theoretical approach. The deep-sea osmolyte trimethylamine-N-oxide is shown to play a crucial and singular role in its ability to not only guarantee sustainability of the native protein's folded state under harsh environmental conditions, but it also controls water-mediated intermolecular interactions at high pressure, thereby preventing contact formation and hence aggregation of proteins.

On the Origin of Microtubules' High-Pressure Sensitivity.

For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.

The effects of osmolytes and crowding on the pressure-induced dissociation and inactivation of dimeric LADH.

Investigating the correlation between structure and activity of oligomeric enzymes at high pressure is essential for understanding intermolecular interactions and reactivity of proteins in cellulo of organisms thriving at extreme environmental conditions as well as for biotechnological applications, such as high-pressure enzymology. In a combined experimental effort employing small-angle X-ray scattering, FT-IR and fluorescence spectroscopy as well as stopped-flow enzyme kinetics in concert with high-pressure techniques, we reveal the pressure-induced conformational changes of the dimeric enzyme horse liver alcohol dehydrogenase (LADH) on the quaternary, secondary and tertiary structural level. Moreover, the effects of cosolutes and crowding agents, mimicking intracellular conditions, have been addressed. Our results show that beyond an increase of enzymatic activity at low pressures, loss of enzyme activity occurs around 600-800 bar, i.e. in a pressure regime where small conformational changes take place in the coenzyme's binding pocket, only. Whereas higher-order oligomers dissociate at low pressures, subunit dissociation of dimeric LADH takes place, depending on the solution conditions, between 2000 and 4000 bar, only. Oligomerization and subunit dissociation are modulated by cosolvents such as urea or trimethylamine-N-oxide as well as by the crowding agent polyethylene glycol, based on their tendency to bind to the protein's interface or act via their excluded volume effect, respectively.

Antibodies under pressure: A Small-Angle X-ray Scattering study of Immunoglobulin G under high hydrostatic pressure.

In the present work two subclasses of the human antibody Immunoglobulin G (IgG) have been investigated by Small-Angle X-ray Scattering under high hydrostatic pressures up to 5kbar. It is shown that IgG adopts a symmetric T-shape in solution which differs significantly from available crystal structures. Moreover, high-pressure experiments verify the high stability of the IgG molecule. It is not unfolded by hydrostatic pressures of up to 5kbar but a slight increase of the radius of gyration was observed at elevated pressures.

Phase behavior of lysozyme solutions in the liquid-liquid phase coexistence region at high hydrostatic pressures.

We present results from small-angle X-ray scattering and turbidity measurements on the effect of high hydrostatic pressure on the phase behavior of dense lysozyme solutions in the liquid-liquid phase separation region, and characterize the underlying intermolecular protein-protein interactions as a function of temperature and pressure under charge-screening conditions (0.5 M NaCl). A reentrant liquid-liquid phase separation region is observed at elevated pressures, which may originate in the pressure dependence of the solvent-mediated protein-protein interaction. A temperature-pressure-concentration phase diagram was constructed for highly concentrated lysozyme solutions over a wide range of temperatures, pressures and protein concentrations including the critical region of the liquid-liquid miscibility gap.

Intramolecular structure and energetics in supercooled water down to 255 K.

We studied the structure and energetics of supercooled water by means of X-ray Raman and Compton scattering. Under supercooled conditions down to 255 K, the oxygen K-edge measured by X-ray Raman scattering suggests an increase of tetrahedral order similar to the conventional temperature effect observed in non-supercooled water. Compton profile differences indicate contributions beyond the theoretically predicted temperature effect and provide a deeper insight into local structural changes. These contributions suggest a decrease of the electron mean kinetic energy by 3.3 ± 0.7 kJ (mol K)(-1) that cannot be modeled within established water models. Our surprising results emphasize the need for water models that capture in detail the intramolecular structural changes and quantum effects to explain this complex liquid.