New Approaches for Studying Disordered States of Proteins by NMR Spectroscopic Methods

Structural and dynamic studies of disordered (i.e. unfolded or partially folded) states of proteins provide important insights into preferential conformations existing these states and into the nature of the free energy landscape of such systems. NMR spectroscopy is a powerful tool capable of determining detailed structural preferences within the unfolded state ensemble, as well as of probing dynamic properties- in solution. The aim of this project is to develop NMR spectroscopic methods for investigating disordered states of proteins, focusing on dynamic properties. These experiments will be applied to an SH3 domain, which exists in slow exchange (on the NMR chemical shift time scale) between a folded state and an unfolded state under non-denaturing conditions, therefore giving rise to two distinctive sets of resonances for the two states. This system thus provides an excellent model for comparative structural and dynamic studies of unfolded and folded states of a protein under identical experimental conditions. The focuses of this project will be the (i) the equilibrium between folded and unfolded states, which can be investigated by several different approaches employing either longitudinal or transverse nuclear spin relaxation, (ii) further studies on microsecond time scale confortri4tional exchange processes within the unfolded state ensemble, which contribute to 15N and 13C line widths, using recently developed NMR spectroscopic methods which determine the dependence of the line broadening on the field strength of an external field and (iii) use of residual dipolar couplings and anisotropic contributions to the chemical shift in aligned media to probe dynamics on the pico- to milisecond time scale.