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) 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 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), 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.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

Trehalose is able to modulate the membrane order and fluidity under disruptive conditions caused by excluded cosolutes or hydrostatic pressure.

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.

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).