Abstract
Recently, it is has been shown that the CN 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 Phe~CN~ 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 Phe~CN~ 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 of calmodulin (CaM) to a peptide derived from the CaM‐binding domain of skeletal muscle myosin light chain kinase (MLCK). The peptide (MLCK~3CN~) contains a single Phe~CN~ residue and has been shown to bind to CaM with high affinity. As expected, addition of CaM into a MLCK~3CN~ solution resulted in quenching of the Phe~CN~ fluorescence. A series of stochiometric titrations further allowed us 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. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 571–576, 2006
This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]