Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP...

Proteomics efforts have created a need for better strategies to functionally categorize newly discovered proteins. To this end, we have employed saturation transfer difference NMR with pools of closely related cofactors, to determine cofactor preferences. This approach works well for dehydrogenases and has also been applied to cyclic nucleotide-binding proteins. In the latter application, a protein (radial spoke protein-2, RSP2) that plays a central role in forming the radial spoke of Chlamydomonas reinhardtii flagella was shown to bind cCMP. cCMP-binding proteins are rare, although previous reports of their presence in sperm and flagella suggest that cCMP may have a more general role in flagellar function. 31P NMR was used to monitor the preferential hydrolysis of ATP versus GTP, suggesting that RSP2 is a kinase.