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A novel fluorescent probe for protein binding and folding studies: p ‐cyano‐phenylalanine
Author(s) -
Tucker Matthew J.,
Oyola Rolando,
Gai Feng
Publication year - 2006
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.20587
Subject(s) - chemistry , fluorescence , fluorophore , calmodulin , peptide , folding (dsp implementation) , quenching (fluorescence) , amino acid , biophysics , phenylalanine , stereochemistry , biochemistry , physics , quantum mechanics , electrical engineering , biology , enzyme , engineering
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 biopolymers@wiley.com