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

Abstract

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow-to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.

NMR Studies of Aromatic Ring Flips to Probe Conformational Fluctuations in Proteins

“…Very recently, we measured ring flip rate constants using 13 C R 1ρ relaxation dispersion caused by residual dipolar coupling (RDC) mediated exchange broadening in weakly aligned proteins. 49 This approach, first demonstrated by the Kay and Palmer groups for 15 N backbone amides and 13 C methyl groups, respectively, 68 , 69 enables measurement of exchange rates even when there is no or very small chemical shift difference between the exchanging sites—which appears to be a relatively common scenario in aromatic side chains, as already mentioned above. The RDC involving the covalently bonded 1 H– 13 C nuclei depends on the orientation of the bond vector with respect to the static magnetic field axis and thus carries powerful structural information.…”

“…The effect of crystal packing on ring flip dynamics in microcrystalline solid-state NMR samples is of potential concern . The detailed intermolecular interactions of exposed aromatic rings can play significant roles in governing ring flip rate constants, ,, but it remains an open question to what extent crystal packing might influence the dynamics of buried rings; apparent discrepancies between solution-state and solid-state NMR data obtained for the small protein GB1 suggest that crystal packing might play a role also in this case. , Further comparative studies are needed in this area.…”