Slow dynamics in folded and unfolded states of an SH3 domain

N-15 relaxation dispersion experiments were applied to the isolated N-termi nal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecon d to second time scale exchange processes. The drkN SH3 domain exists in eq uilibrium between folded (F-exch) and unfolded (U-exch) states under nonden aturing conditions in a ratio of 2:1 at 20 degreesC, with an average exchan ge rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical sh ift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U-exch ensemble there is a contiguous stretch of residues undergoing conformational exchange on a mus/ms time sc ale, likely due to local, non-native hydrophobic collapse. For these residu es both the F-exch <----> U-exch conformational. exchange process and the m us/ms exchange event within the U-exch state contribute to the 15N line wid th and can be analyzed using CPMG-based 15N relaxation dispersion measureme nts. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental 15N relaxation d ispersion data with results from an 15N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farro w, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the depe ndence of the effective transverse relaxation rate constant on the pulse sp acing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that re laxation dispersion experiments provide an extremely sensitive tool to prob e conformational exchange processes in unfolded states and to obtain inform ation on the free energy landscape of such systems.