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Zusammenfassung:
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 in a protein can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy called methionine scanning that overcomes the aforementioned problems of methyl NMR spectroscopy. We systematically substitute solvent-exposed residues with reporter methionines in the expected binding site and perform chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. Using these mutants, we can instantaneously assign all newly arising reporter methyl signals, determine interaction surfaces on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. We have applied our methionine scanning methodology to a number of protein complexes including high-molecular weight systems such as the 11S activator-proteasome complex. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl NMR studies.
