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

Erwin Schrödinger Fellowship J 1933 New NMR spectroscopic methods for disordered proteins Martin TOLLINGER 26.6.2000 Structural and dynamic studies of disordered (i.e. unfolded or partially folded) states of proteins provide important insights into and any preferential conformations existing in these states and into the initiation of protein folding. Experimental NMR spectroscopy is a powerful tool capable of generating detailed site-specific evidence for structural preferences within the unfolded state ensemble, as well as of dynamic properties of these molecules in solution. The aim of this project is to develop new NMR spectroscopic methods for investigating disordered states of proteins, focusing on residual dipolar coupling data. While, in recent years, residual dipolar couplings have been employed to obtain structural information on folded proteins, no dipolar coupling measurements on disordered states of proteins have been reported in the literature. In this project, dipolar coupling s will be used i) to extract information on the dynamic properties of peptide planes in terms of generalized degree of order parameters and ii) to determine the time-averaged orientation of the internuclear vectors which correspond to the coupling partners within the unfolded state ensemble. The second part of the project is the investigation of backbone amide proton exchange rate in an unfolded protein using NNM relaxation measurements of multispin order. Backbone amide proton exchange provides interesting structural information on residual local structure. These NMR spectroscopic methods will be applied to the N-terminal SH3 domain of the Drosophila signal transduction protein drk, which exists in an equilibrium between a folded and unfolded states under non-denaturing conditions. This system, which has been investigated extensively, represents an ideal model system for the development of novel NMR spectroscopic methods for disordered states of proteins, enabling comparison to other, previosly determined.