title: Utilization of lysine ¹³C-methylation NMR for protein-protein interaction studies.
abstract: Chemical modification is an easy way for stable isotope labeling of non-labeled proteins. The reductive (13)C-methylation of the amino group of the lysine side-chain by (13)C-formaldehyde is a post-modification and is applicable to most proteins since this chemical modification specifically and quickly proceeds under mild conditions such as 4 °C, pH 6.8, overnight. (13)C-methylation has been used for NMR to study the interactions between the methylated proteins and various molecules, such as small ligands, nucleic acids and peptides. Here we applied lysine (13)C-methylation NMR to monitor protein-protein interactions. The affinity and the intermolecular interaction sites of methylated ubiquitin with three ubiquitin-interacting proteins were successfully determined using chemical-shift perturbation experiments via the (1)H-(13)C HSQC spectra of the (13)C-methylated-lysine methyl groups. The lysine (13)C-methylation NMR results also emphasized the importance of the usage of side-chain signals to monitor the intermolecular interaction sites, and was applicable to studying samples with concentrations in the low sub-micromolar range.
authors.affiliation: Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Hattori
authors.forename: Kyoko
authors.initials: K
authors.lastname: Furuita
authors.forename: Izuru
authors.initials: I
authors.lastname: Ohki
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.forename: Harumi
authors.initials: H
authors.lastname: Fukada
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shirakawa
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Chojiro
authors.initials: C
authors.lastname: Kojima
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D008745:Methylation; D062105:Molecular Docking Simulation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D011506:Proteins; D025801:Ubiquitin; D010450:Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase; D008239:Lysine