We present results from small-angle x-ray scattering data on the effect of high 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 in this region of phase space. A reentrant liquid-liquid phase separation region has been discovered at elevated pressures, which originates in the pressure dependence of the solvent-mediated protein-protein interactions.

Understanding the intermolecular interaction potential, V(r), of proteins under the influence of temperature, pressure, and salt concentration is essential for understanding protein aggregation, crystallization, and protein phase behavior in general. Here, we report small-angle x-ray scattering studies on dense lysozyme solutions of high ionic strength as a function of temperature and pressure. We show that the interaction potential changes in a nonlinear fashion over a wide range of temperatures, salt, and protein concentrations. Neither temperature nor protein and salt concentration lead to marked changes in the pressure dependence of V(r), indicating that changes of the water structure dominate the pressure dependence of the intermolecular forces. Furthermore, by analysis of the temperature, pressure, and ionic strength dependence of the normalized second virial coefficient, b2, we show that the interaction can be fine-tuned by pressure, which can be used to optimize b2 values for controlled protein crystallization.

FT-IR spectroscopic, small-angle X-ray scattering and calorimetric measurements have been applied to explore the effect of the macromolecular crowder agent Ficoll on the temperature- and pressure-dependent stability diagram and folding reaction of the protein Staphylococcal Nuclease (SNase). Additionally, we compare the experimental data with approximate theoretical predictions. We found that temperature- and pressure-induced equilibrium unfolding of SNase is markedly shifted to higher temperatures and pressures in 30 wt% Ficoll solutions. The structure of the unfolded state ensemble does not seem to be strongly influenced in the presence of the crowder. Self-crowding effects have been found to become important at SNase concentrations above 10 wt% only. Our kinetic results show that the folding rate of SNase decreases markedly in the presence of Ficoll. These results indicate that besides the commonly encountered excluded volume effect, other factors need to be considered when assessing confinement effects on protein folding kinetics. Among those, crowder-induced viscosity changes seem to be prominent.

We investigated thermosensitive poly(N-isopropylacrylamide) microgels by high-pressure small angle X-ray scattering and Fouriertransform infrared spectroscopy below and above the collapse temperature. The measurements reveal little pressure-induced deswelling below the volume phase transition temperature and clear re-swelling of the collapsed gels at temperatures above the VPTT.

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