Osmolytes modify protein dynamics and function of tetrameric lactate dehydrogenase upon pressurization.

We present a study of the combined effects of natural cosolvents (TMAO, glycine, urea) and pressure on the activity of the tetrameric enzyme lactate dehydrogenase (LDH). To this end, high-pressure stopped-flow methodology in concert with fast UV/Vis spectroscopic detection of product formation was applied. To reveal possible pressure effects on the stability and dynamics of the enzyme, FTIR spectroscopic and neutron scattering measurements were carried out. In neat buffer solution, the catalytic turnover number of the enzyme, kcat, increases up to 1000 bar, the pressure range where dissociation of the tetrameric species to dimers sets in. Accordingly, we obtain a negative activation volume, ΔV# = -45.3 mL mol-1. Further, the enzyme substrate complex has a larger volume compared to the enzyme and substrate in the unbound state. The neutron scattering data show that changes in the fast internal dynamics of the enzyme are not responsible for the increase of kcat upon compression. Whereas the magnitude of kcat is similar in the presence of the osmolytes, the pressure of deactivation is modulated by the addition of cosolvents. TMAO and glycine increase the pressure of deactivation, and in accordance with the observed stabilizing effect both cosolvents exhibit against denaturation and/or dissociation of proteins. While urea does not markedly affect the magnitude of the Michaelis constant, KM, both 1 M TMAO and 1 M glycine exhibit smaller KM values of about 0.07 mM and 0.05 mM below about 1 kbar. Such positive effect on the substrate affinity could be rationalized by the effect the two cosolutes impose on the thermodynamic activities of the reactants, which reflect changes in water-mediated intermolecular interactions. Our data show that the intracellular milieu, i.e., the solution conditions that have evolved, may be sufficient to maintain enzymatic activity under extreme environmental conditions, including the whole pressure range encountered on Earth.

Combined effects of osmotic and hydrostatic pressure on multilamellar lipid membranes in the presence of PEG and trehalose.

We studied the interaction of lipid membranes with the disaccharide trehalose (TRH), which is known to stabilize biomembranes against various environmental stress factors. Generally, stress factors include low/high temperature, shear, osmotic and hydrostatic pressure. Small-angle X-ray-scattering was applied in combination with fluorescence spectroscopy and calorimetric measurements to get insights into the influence of trehalose on the supramolecular structure, hydration level, and elastic and thermodynamic properties as well as phase behavior of the model biomembrane DMPC, covering a large region of the temperature, osmotic and hydrostatic pressure phase space. We observed distinct effects of trehalose on the topology of the lipid's supramolecular structure. Trehalose, unlike osmotic pressure induced by polyethylene glycol, leads to a decrease of lamellar order and a swelling of multilamellar vesicles, which is attributable to direct interactions between the membrane and trehalose. Our results revealed a distinct biphasic concentration dependence of the observed effects of trehalose. While trehalose intercalates between the polar head groups at low concentrations, the effects after saturation are dominated by the exclusion of trehalose from the membrane surface.

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.