ABSTRACTProteins often interconvert between different conformations in ways critical to their function. While manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic beta-strand slip as it switches between two stably-folded conformations. Coupling high pressure and biomolecular NMR, we obtained the first quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wildtype equivalent, and the interconversion proceeds through a chiefly-unfolded intermediate state. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.
High-Pressure X-ray Protein Crystallography
