title: Methionine scanning as an NMR tool for detecting and analyzing biomolecular interaction surfaces.
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.
authors.affiliation: Max Planck Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany.
authors.forename: Mira C
authors.initials: MC
authors.lastname: Stoffregen
authors.forename: Matthias M
authors.initials: MM
authors.lastname: Schwer
authors.forename: Fabian A
authors.initials: FA
authors.lastname: Renschler
authors.forename: Silke
authors.initials: S
authors.lastname: Wiesner
mesh_terms: D001665:Binding Sites; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D008715:Methionine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D020033:Protein Isoforms; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D008715:Methionine; C501306:Ubc4 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae