Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations

Abstract

Proteins are dynamic entities that intermittently depart from their ground-state structures and undergo conformational transitions as a critical part of their functions. Central to understanding such transitions are the structural rearrangements along the connecting pathway, where the transition state plays a special role. Using NMR relaxation at variable temperature and pressure to measure aromatic ring flips inside a protein core, we obtain information on the structure and thermodynamics of the transition state. We show that the isothermal compressibility coefficient of the transition state is similar to that of short-chain hydrocarbon liquids, implying extensive local unfolding of the protein. Our results further indicate that the required local volume expansions of the protein can occur not only with a net positive activation volume of the protein, as expected from previous studies, but also with zero activation volume by compaction of remote void volume, when averaged over the ensemble of states.

Visualizing protein breathing motions associated with aromatic ring flipping

“…4e, f ). This cavity expansion is in agreement with previous studies that have reported activation volumes between 40 and 85 Å 3 for ring flipping events of aromatic residues in other proteins 12 , 14 , 21 , 40 , 41 . The expansion is followed by a compaction of the surrounding protein environment as the ring becomes stabilized by CH–π interactions from L519 (Fig.…”

“…Breathing motions along the structural trajectory between the major and the minor state generate the necessary void volume for fast ring flipping of Y526 to take place (Supplementary Video 2 ). Although a recent NMR study suggested extensive local unfolding as the source of cavity creation 41 , our study provides an alternative view by showing how a substantial void volume can be generated through distinct structural rearrangements, while maintaining the overall protein fold.…”

Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms

“…We have previously determined the activation parameters of the F52 ring flip, including Δ H ‡ and Δ S ‡ , as well as the activation volume, Δ V ‡ , and the isothermal volume compressibility . These parameters present a consistent picture of the transition state as being significantly expanded with fewer stabilizing interactions than the ground state, while populating a larger number of conformations and having a compressibility similar to that of unfolded proteins, indicating that the F52 ring flip is made possible by relatively large-scale breathing motions that resemble local unfolding .…”

“…Aromatic ring flips, i.e., 180° rotations of the χ 2 dihedral angle in Phe and Tyr side chains, are a hallmark of transient conformational fluctuations in proteins. , Aromatic side chains in the interior of globular proteins commonly undergo rapid rotations, as gauged by the observation of a single peak in the NMR spectrum originating from the two symmetry related nuclei of the ring. In the past, ring flip rates have been measured for only a limited number of proteins by using line shape analysis or longitudinal exchange experiments. ,− The development of site-selective isotope labeling schemes for aromatic side chains − has enabled relaxation dispersion experiments for aromatic 13 C and 1 H sites − and led to a renaissance in studying ring flips by NMR spectroscopy. − …”

“…The rotation of an aromatic side chain located inside the protein core requires that the surrounding side chains transiently create sufficient volume to accommodate the ring rotation. Temperature- and pressure-dependent experiments make it possible to determine the activation enthalpy (Δ H ‡ ), entropy (Δ S ‡ ), and volume (Δ V ‡ ) of this process. ,,,,, Recently, we presented a combined analysis of temperature- and pressure-dependent ring flip data that enabled us to also determine the change in compressibility (Δ κ ‡ ) between ground and transition states of a ring flip, which indicates that the transition state is liquidlike, whereas the ground state is solidlike. This result differs from conclusions reached in another study of the dynamics of surface-exposed aromatic residues in ubiquitin, which suggest that the ground state is liquidlike and the aromatic rings undergo continuous diffusive rotations .…”