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Reference: Stoffregen MC, et al. (2012) Methionine scanning as an NMR tool for detecting and analyzing biomolecular interaction surfaces. Structure 20(4):573-81

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Abstract


Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.CI - Copyright (c) 2012 Elsevier Ltd. All rights reserved.

Reference Type
Journal Article | Research Support, Non-U.S. Gov't
Authors
Stoffregen MC, Schwer MM, Renschler FA, Wiesner S
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