NMR-based screening of proteins containing 13C-labeled methyl groups

A method is described for NMR-based screening that involves monitoring the C-13/H-1 chemical shift changes of a protein selectively labeled with C-13 at the methyl groups of valine, leucine, and isoleucine (delta 1 only). Using this approach, the sensitivity is increased by nearly 3-fold compared with that of NMR-based screening using H-1/N-15 chemical shifts. A synthetic route is described for the inexpensive production of the labeled amino acid precursors [3,3'-C-13]-alpha-ketoisovalerate and [3-C-13]-alpha-ketobutyrate, making the cost of protein preparation comparable to that of uniform N-15 labeling. In addition to enhancing the NMR-based screening efforts directed against low molecular weight proteins (MW less than or equal to 30 kDa), the use of the selective methyl labels in combination with deuterium labeling is advantageous for screening high molecular weight protein targets (MW greater than or equal to 100 MDa).