title: Effect of molecular crowding on the temperature-pressure stability diagram of ribonuclease A.
abstract: FT-IR spectroscopic and thermodynamic measurements were designed to explore the effect of a macromolecular crowder, dextran, on the temperature and pressure-dependent phase diagram of the protein Ribonuclease A (RNase A), and we compare the experimental data with approximate theoretical predictions based on configuration entropy. Exploring the crowding effect on the pressure-induced unfolding of proteins provides insight in protein stability and folding under cell-like dense conditions, since pressure is a fundamental thermodynamic variable linked to molecular volume. Moreover, these studies are of relevance for understanding protein stability in deep-sea organisms, which have to cope with pressures in the kbar range. We found that not only temperature-induced equilibrium unfolding of RNase A, but also unfolding induced by pressure is markedly prohibited in the crowded dextran solutions, suggesting that crowded environments such as those found intracellularly, will also oppress high-pressure protein unfolding. The FT-IR spectroscopic measurements revealed a marked increase in unfolding pressure of 2 kbar in the presence of 30 wt % dextran. Whereas the structural changes upon thermal unfolding of the protein are not significantly influenced in the presence of the crowding agent, through stabilization by dextran the pressure-unfolded state of the protein retains more ordered secondary structure elements, which seems to be a manifestation of the entropic destabilization of the unfolded state by crowding.
authors.affiliation: Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry, TU Dortmund University, Germany.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhai
authors.forename: Roland
authors.initials: R
authors.lastname: Winter
mesh_terms: D003911:Dextrans / Q000737:chemistry*; D011312:Pressure; D058767:Protein Unfolding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D003911:Dextrans; D012259:Ribonuclease, Pancreatic