Using Nitrile-Derivatized Amino Acids as Infrared Probes of Local Environment

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Abstract
It is well-known that the C⋮N stretching vibration in acetonitrile is sensitive to solvent. Therefore, we proposed in this contribution to use this vibrational mode to report local environment of a particular amino acid in proteins or local environmental changes upon binding or folding. We have studied the solvent-induced frequency shift of two nitrile-derivatized amino acids, which are, AlaCN and PheCN, in H2O and tetrahydrofuran (THF), respectively. Here, THF was used to approximate a protein's hydrophobic interior because of its low dielectric constant. As expected, the C⋮N stretching vibrations of both AlaCN and PheCN shift as much as ∼10 cm-1 toward higher frequency when THF was replaced with H2O, indicative of the sensitivity of this vibration to solvation. To further test the utility of nitrile-derivatized amino acids as probes of the environment within a peptide, we have studied the binding between calmodulin (CaM) and a peptide from the CaM binding domain of skeletal muscle myosin light chain kinase (MLCK579-595), which contains a single PheCN. MLCK579-595 binds to CaM in a helical conformation. When the PheCN was substituted on the polar side of the helix, which was partially exposed to water, the C⋮N stretching vibration is similar to that of PheCN in water. In constrast, when PheCN is introduced at a site that becomes buried in the interior of the protein, the C⋮N stretch is similar to that of PheCN in THF. Together, these results suggest that the C⋮N stretching vibration of nitrile-derivatized amino acids can indeed be used as local internal environmental markers, especially for protein conformational studies.

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