A novel fluorescent probe for protein binding and folding studies:: p-cyano-phenylalanine

Recently, it is has been shown that the C=N stretching vibration of a non-natural amino acid, p-cyano-phenylalanine (Phe(CN)), could be used as an infrared reporter of local environment. Here. we further showed that the fluorescence emission of PheCN is also sensitive to solvent and, therefore, could be used as a novel optical probe for protein binding and folding studies. Moreover, we found that the fluorescence quantum yield of PheCN is nearly five times larger than that of phenylalanine and, more importantly, can be selectively excited even when other aromatic amino acids are present, thus making it a more versatile fluorophore. To test the feasibility of using Phe(CN) as a practical fluorescent probe, we studied the binding 4 calmodulin (CaM) to a peptide derived from the CaM-binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK3CN) contains a single Phe(CN) residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK3CN solution resulted in quenching of the Phe(CN) fluorescence. A series of stochiometric titrations further allowed its to determine the binding affinity (K-d) of this peptide to CaM. Taken together, these results indicated that the Phe(CN) fluorescence is sensitive to environment and could be applicable to a wide variety, of biological problems. (c) 2006 Wiley Periodicals, Inc.