Yoshikazu Hattori, Kyoko Furuita, Izuru Ohki, Takahisa Ikegami, Harumi Fukada, Masahiro Shirakawa, Toshimichi Fujiwara, Chojiro Kojima
Journal of biomolecular NMR 2013 JanChemical 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.
Yoshikazu Hattori, Kyoko Furuita, Izuru Ohki, Takahisa Ikegami, Harumi Fukada, Masahiro Shirakawa, Toshimichi Fujiwara, Chojiro Kojima. Utilization of lysine ¹³C-methylation NMR for protein-protein interaction studies. Journal of biomolecular NMR. 2013 Jan;55(1):19-31
PMID: 23224986
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