Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study

Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial ¹⁵ N-, ¹³ C^α -, and ¹³ C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves ¹⁵ N, 1-¹³ C, 2-¹³ C, fully ¹⁵ N/¹³ C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide ¹ H and ¹⁵ N resonances need to be obtained, even in cases where sensitivity is the limiting factor.