A method is described for NMR-based screening that involves monitoring the 13C/1H chemical shift changes of a protein selectively labeled with 13C at the methyl groups of valine, leucine, and isoleucine (δ1 only). Using this approach, the sensitivity is increased by nearly 3-fold compared with that of NMR-based screening using 1H/15N chemical shifts. A synthetic route is described for the inexpensive production of the labeled amino acid precursors [3,3'-13C]-α-ketoisovalerate and [3-13C]-α-ketobutyrate, making the cost of protein preparation comparable to that of uniform 15N labeling. In addition to enhancing the NMR-based screening efforts directed against low molecular weight proteins (MW ≤ 30 kDa), the use of the selective methyl labels in combination with deuterium labeling is advantageous for screening high molecular weight protein targets (MW ≥ 100 kDa).
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry