Slow Dynamics in Folded and Unfolded States of an SH3 Domain

Abstract

¹⁵N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F_exch) and unfolded (U_exch) states under nondenaturing conditions in a ratio of 2:1 at 20 °C, with an average exchange rate constant, k_ex, of 2.2 s^-1 (slow exchange on the NMR chemical shift 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 μs/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F_exch ↔ U_exch conformational exchange process and the μs/ms exchange event within the U_exch state contribute to the ¹⁵N line width and can be analyzed using CPMG-based ¹⁵N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental ¹⁵N relaxation dispersion data with results from an ¹⁵N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR1994, 4, 727−734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing 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 relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.