期刊
JOURNAL OF BIOMOLECULAR NMR
卷 75, 期 6-7, 页码 221-232出版社
SPRINGER
DOI: 10.1007/s10858-021-00370-0
关键词
NMR; Assignment; Methyl groups; Acetolactate; HSP90
资金
- FRISBI [ANR-10-INBS-05-02]
- GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS [ANR-17-EURE-0003]
- CEA/NMR-Bio [C24990]
- French National Research Agency [ANR-15-IDEX-02]
The study presents a new synthetic precursor for connecting leucine and valine pro-S methyl moieties to backbone atoms, facilitating quantitative NMR studies of large proteins. This optimized precursor allows for direct transfer of backbone assignment to corresponding methyl groups without correction of H-1/H-2 isotopic shifts on C-13 resonances, as demonstrated on the N-terminal domain of human HSP90.
Methyl moieties are highly valuable probes for quantitative NMR studies of large proteins. Hence, their assignment is of the utmost interest to obtain information on both interactions and dynamics of proteins in solution. Here, we present the synthesis of a new precursor that allows connection of leucine and valine pro-S methyl moieties to backbone atoms by linear C-13-chains. This optimized H-2/C-13-labelled acetolactate precursor can be combined with existing C-13/H-2-alanine and isoleucine precursors in order to directly transfer backbone assignment to the corresponding methyl groups. Using this simple approach leucine and valine pro-S methyl groups can be assigned using a single sample without requiring correction of H-1/H-2 isotopic shifts on C-13 resonances. The approach was demonstrated on the N-terminal domain of human HSP90, for which complete assignment of Ala-beta, Ile-delta(1), Leu-delta(2), Met-epsilon, Thr-gamma and Val-gamma(2) methyl groups was obtained.
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