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.

A high pressure study of calmodulin-ligand interactions using small-angle X-ray and elastic incoherent neutron scattering.

Calmodulin (CaM) is a Ca2+ sensor and mediates Ca2+ signaling through binding of numerous target ligands. The binding of ligands by Ca2+-saturated CaM (holo-CaM) is governed by attractive hydrophobic and electrostatic interactions that are weakened under high pressure in aqueous solutions. Moreover, the potential formation of void volumes upon ligand binding creates a further source of pressure sensitivity. Hence, high pressure is a suitable thermodynamic variable to probe protein-ligand interactions. In this study, we compare the binding of two different ligands to holo-CaM as a function of pressure by using X-ray and neutron scattering techniques. The two ligands are the farnesylated hypervariable region (HVR) of the K-Ras4B protein, which is a natural binding partner of holo-CaM, and the antagonist trifluoperazine (TFP), which is known to inhibit holo-CaM activity. From small-angle X-ray scattering experiments performed up to 3000 bar, we observe a pressure-induced partial unfolding of the free holo-CaM in the absence of ligands, where the two lobes of the dumbbell-shaped protein are slightly swelled. In contrast, upon binding TFP, holo-CaM forms a closed globular conformation, which is pressure stable at least up to 3000 bar. The HVR of K-Ras4B shows a different binding behavior, and the data suggest the dissociation of the holo-CaM/HVR complex under high pressure, probably due to a less dense protein contact of the HVR as compared to TFP. The elastic incoherent neutron scattering experiments corroborate these findings. Below 2000 bar, pressure induces enhanced atomic fluctuations in both holo-CaM/ligand complexes, but those of the holo-CaM/HVR complex seem to be larger. Thus, the inhibition of holo-CaM by TFP is supported by a low-volume ligand binding, albeit this is not associated with a rigidification of the complex structure on the sub-ns Å-scale.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

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.

Building Polyelectrolyte Multilayers with Calmodulin: A Neutron and X-ray Reflectivity Study.

We have studied the formation and functional properties of polyelectrolyte multilayers where calmodulin (CaM) is used as a polyanion. CaM is known to populate distinct conformational states upon binding Ca2+ and small ligand molecules. Therefore, we have also probed the effects of Ca2+ ions and trifluoperazine (TFP) as ligand molecule on the interfacial structures. Multilayers with the maximum sequence PEI-(PSS-PAH)x-CaM-PAH-CaM-PAH have been deposited on silicon wafers and characterized by X-ray and neutron reflectometry. From the analysis of all data, several remarkable conclusions can be drawn. When CaM is deposited for the second time, a much thicker sublayer is produced than in the first CaM deposition step. However, upon rinsing with PAH, very thin CaM-PAH sublayers remain. There are no indications that ligand TFP can be involved in the multilayer buildup due to strong CaM-PAH interactions. However, there is a significant increase in the multilayer thickness upon removal of Ca2+ ions from holo-CaM and an equivalent decrease in the multilayer thickness upon subsequent saturation of apo-CaM with Ca2+ ions. Presumably, CaM can still be toggled between an apo and a holo state, when it is embedded in polyelectrolyte multilayers, providing an approach to design bioresponsive interfaces.

Near-Surface and Bulk Behavior of Bicontinuous Microemulsions under High-Pressure Conditions.

The effect of hydrostatic pressure on the structure of a bicontinuous microemulsion in the presence of a solid interface has been studied by X-ray reflectometry and compared to the bulk behavior determined by small-angle X-ray scattering. Surface-induced lamellar ordering is observed close to the hydrophilic interface, which persists upon compression. The lamellar domains are compressed, but the correlation length of lamellar order does not change with pressure. SAXS measurements on the bulk microemulsion revealed an increased order upon pressurization. Although pressure can cause the formation of highly ordered lamellar phases from ordered bicontinuous cubic phases, such a scenario is not observed for the disordered analogue studied here. High pressure increases the stiffness of the interfacial surfactant layer, but this is not sufficient to overcome the loss in conformational entropy that would result from a transition to an ordered lamellar phase. Possible technological and biological implications of our results are briefly discussed.