Journal
CHEMISTRYSELECT
Volume 5, Issue 17, Pages 5092-5098Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/slct.202000827
Keywords
NMR; asymmetric synthesis; Threonine; Methyl; Isotopic labelling; Large proteins; Distance restraints; nOes
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Funding
- 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 in the framework of the Investissements d'avenir program [ANR-15-IDEX-02]
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Methyl groups are valuable probes for solution NMR, allowing the investigation of large protein complexes. Among the six methyl containing residues, threonine has one of the less hydrophobic side chain, and can reside both within the interior of a protein or on the protein surface. This article presents an efficient mixed chemical/enzymatic synthesis scheme, enabling the preparation of threonine with natural configuration on the two stereogenic centers together with an optimal introduction of H-1/H-2 and C-12/C-13 atoms at specific sites. Such specifically labelled amino acid can be efficiently incorporated in overexpressed proteins without scrambling or in combination with any other types of (CH3)-C-13 probes. Additionally we report application to the 82 kDa Malate Synthase G. Our findings demonstrate that structural meaningful long range nOes can be detected between threonine methyl probes and methionine and isoleucine methyl groups distant by 12 angstrom.
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