Selective Excitation of Cyanophenylalanine Fluorophores for Multi-Site Binding Studies

Recently, it has been shown that nitrile-derivatized phenylalanines possess distinct fluorescent properties depending on the position of the cyano-group within the aromatic ring. These fluorophores have potential as probes for studying protein dynamics due to their sensitivity to local environment. Herein, we demonstrate that 2-cyanophenylalanine (Phe_2CN) and Phe_4CN can independently monitor multiple sites during the Ca²⁺ dependent binding of a skeletal muscle myosin light chain kinase (MLCK) peptide fragment to the protein calmodulin (CaM). These cyano-probes were incorporated at two different positions along the peptide chain and monitored simultaneously via selective excitation of the two chromophores. The peptide was labeled with Phe_4CN at a residue known to bind to a hydrophobic binding pocket of CaM, while Phe_2CN was designed to acquire dynamics external to the binding pocket. By selectively exciting each of the chromophores, it was determined that the fluorescence emission of Phe_4CN located at position 581 of MLCK was quenched in the presence of CaM, while no significant change in Phe_2CN emission was observed at exposed position 594. The CaM binding affinity (K_d) of the double labeled MLCK peptide was calculated to be approximately 64 nM, which is in agreement with previous measurements. These results indicate that multiple Phe_CN reporters within the same peptide can simultaneously detect variations in the local environment, and that these fluorophores could be utilized to investigate a wide variety of biological problems.