NMR Relaxation Dispersion Methods for the Structural and Dynamic Analysis of Quickly Interconverting, Low-Populated Conformational Substates.

Most biomolecular processes involve proteins shuttling among different conformational states, particularly from highly populated ground states to the lowly populated excited states that determine the interconversion rates and biological function, and which are invisible to most structural biology techniques. These structural transitions are rare and relatively fast: happen in the millisecond-microsecond timescale (ms-μs). NMR spectroscopy can access these timescales via relaxation dispersion techniques (RD-NMR). The exchange parameters extracted from RD-NMR experiments provide pivotal information on these otherwise invisible states that reports on key properties of the high free energy, reactive regions of the protein's energy landscape, including the mechanisms of folding/unfolding and of the interconversion between active and inactive states. Here, we describe a simple, step-by-step protocol to carry out RD-NMR experiments on proteins to detect the existence of such conformational substates and characterize their structural properties (chemical shifts).