Time-resolved Solid-state Nmr: Small Molecules and Enzymes in Rapidly Frozen Solution

An important aspect of understanding enzymatic reaction mechanisms is the determination of the molecular structures of enzyme-bound substrates and products. NMR spectroscopy is in a unique position in that it is the only relatively high resolution structural technique which can focus on specific parts of the molecule, such as enzyme-bound intermediates. It has been widely applied to the study of enzyme mechanisms in both the solution and solid states [1-3] at ambient temperatures and at sub-zero temperatures [4]. However, the current molecular weight limit in solution is around 50 kDa [2]. In contrast, with solid-state NMR spectroscopy [5] anisotropic and dipolar broadening can be reduced by cross-polarization magic angle sample spinning (CP-MASS), and there is no known molecular weight limit. We have therefore sought to develop a new technique called time-resolved solid-state NMR spectroscopy [6] which involves applying CP-MASS NMR to studying rapidly freeze-quenched enzyme-substrate mixtures.