Pressure-Dependent Aromatic Ring Flips Reveal Variable Transition-State Volume and Compressibility Among Structural Regions of BPTI.

Conformational transitions are critical for protein function and often involve substantial rearrangements of the interior packing of amino acid residues. Rotations of aromatic side chains-so called "ring flips"-require transient expansion of the surrounding protein core and represent hallmark examples of transient conformational fluctuations. We recently demonstrated that activation volume and isothermal compressibility provide unique insights into ring flip transition states. Here, we report transition-state compressibilities for four aromatic side chains in bovine pancreatic trypsin inhibitor (BPTI). The compressibilities vary widely, ranging from values typical of unfolded proteins to those of the fully folded state. Notably, transition-state compressibility correlates negatively with activation energy, indicating that the likelihood of a ring flip scales with the degree of expansion in the transition state. Our results show that different structural regions, even within a small protein such as BPTI, display distinct dynamic transitions and volume fluctuations, reflecting varying extents of structural rearrangement along the transition pathways. Thus, isothermal compressibility emerges as a powerful parameter yielding insights into the relationships between ground-state structure and the transition-state expansion during conformational transitions.