Measurement of methyl group motional parameters of invisible, excited protein states by NMR spectroscopy.

An understanding of many biological processes can only be achieved through studies of the structure (enthalpy) and motions (entropy) of the key molecules that are involved, including those that are formed only transiently and with low population. These transiently formed, low populated states are invisible to most biophysical techniques but in many cases they can be studied in detail using relaxation dispersion NMR spectroscopy. Relaxation dispersion methodology has recently been described for the measurement of protein backbone excited state chemical shifts as well as bond vector orientations, which form the basis for structural studies of these invisible conformers. It is of interest to extend such studies by quantifying motional parameters of the excited state, providing a more complete description of the energy landscape that drives the biochemical event in question. Herein we describe a relaxation dispersion method for measuring site-specific motional parameters of methyl containing residues in the excited state. The approach is applied to the invisible unfolded state of the G48M Fyn SH3 domain that is in exchange with the folded conformation. Not surprisingly, the degree of disorder is in general higher in the unfolded state than in the folded conformer, although there is some ordering of side-chains in the unfolded state toward the C-terminal region of the domain. The development of the present methodology provides the first step toward characterizing the motional properties of invisible conformers, complementing the structural information that is already available from relaxation dispersion studies.