Ring flips revisited: (13)C relaxation dispersion measurements of aromatic side chain dynamics and activation barriers in basic pancreatic trypsin inhibitor.

Intramolecular motions of proteins are critical for biological function. Transient structural fluctuations underlie a wide range of processes, including enzyme catalysis, ligand binding to buried sites, and generic protein motions, such as 180° rotation of aromatic side chains in the protein interior, but remain poorly understood. Understanding the dynamics and molecular nature of concerted motions requires characterization of their rates and energy barriers. Here we use recently developed (13)C transverse relaxation dispersion methods to improve our current understanding of aromatic ring flips in basic pancreatic trypsin inhibitor (BPTI). We validate these methods by benchmarking ring-flip rates against the three previously characterized cases in BPTI, namely, Y23, Y35, and F45. Further, we measure conformational exchange for one additional aromatic ring, F22, which can be interpreted in terms of a flip rate of 666 s(-1) at 5 °C. Upon inclusion of our previously reported result that Y21 also flips slowly [Weininger, U., et al. (2013) J. Phys. Chem. B 117, 9241-9247], the (13)C relaxation dispersion experiments thus reveal relatively slow ring-flip rates for five of eight aromatic residues in BPTI. These results are in contrast with previous reports, which have estimated that all rings, except Y23, Y35, and F45, flip with a high rate at ambient temperature. The (13)C relaxation dispersion data result in an updated rank order of ring-flip rates in BPTI, which agrees considerably better with that estimated from a recent 1 ms molecular dynamics trajectory than do previously published NMR data. However, significant quantitative differences remain between experiment and simulation, in that the latter yields flip rates that are in many cases too fast by 1-2 orders of magnitude. By measuring flip rates across a temperature range of 5-65 °C, we determined the activation barriers of ring flips for Y23, Y35, and F45. Y23 and F45 have identical activation parameters, suggesting that the fluctuations of the protein core around these residues are similar in character. Y35 differs from the other two in its apparent activation entropy. These results might be rationalized by the fact that Y23 and F45 are located in the same region of the structure while Y35 is remote from the other two rings. As indicated by our new results for the exceptionally well-characterized protein BPTI, (13)C relaxation dispersion experiments open the possibility of studying ring flips in a range of cases wider than that previously possible.