Slow Aromatic Ring Flips Detected Despite Near-Degenerate NMR Frequencies of the Exchanging Nuclei

Aromatic ring flips of Phe and Tyr residues are a hallmark of protein dynamics with a long history in molecular biophysics. Ring flips lead to symmetric exchange of nuclei between sites with distinct magnetic environments, which can be probed by NMR spectroscopy. Current knowledge of ring-flip rates originates from rare cases in which the chemical shift difference between the two sites is sufficiently large and the ring-flip rate sufficiently slow, typically kflip < 10(3) s(-1), so that separate peaks are observed in the NMR spectrum for the two nuclei, enabling direct measurement of the flip rate. By contrast, a great majority of aromatic rings show single peaks for each of the pairs of δ or ε nuclei, which commonly are taken as inferential evidence that the flip rate is fast, kflip < 10(3) s(-1), even though rate measurements have not been achieved. Here we report a novel approach that makes it possible to identify slow ring flips in previously inaccessible cases where only single peaks are observed. We demonstrate that Y21 in the bovine pancreatic trypsin inhibitor (BPTI) has a slow ring-flip rate, kflip < 100 s(-1), a result that contrasts with previous estimates of 10(4)-10(6) s(-1) inferred from the single-peak spectrum of Y21. Comparison with a recent 1 ms molecular dynamics trajectory of BPTI shows qualitative agreement and highlights the value of accurate aromatic ring flip data as an important benchmark for molecular dynamics simulations of proteins across wide time scales.