Expanding the Utility of 4‐cyano‐L‐phenylalanine as a Vibrational Reporter

Abstract

Unnatural amino acids (UAAs) have become useful tools for understanding protein structure and local environments. One of the most commonly used vibrational reporter UAAs to study local protein hydration environments is 4‐cyano‐L‐phenylalanine (pCNF) due to the position and sensitivity of the nitrile symmetric stretching frequency as well as the ability to efficiently incorporate this UAA site‐specifically into proteins using the Amber codon suppression methodology. Additionally, pCNF provides promise to function as a distance reporter when coupled with two‐dimensional infrared (2D IR) spectroscopy. Here, a number of superfolder green fluorescent protein (sfGFP) constructs have been generated with pCNF site‐specifically incorporated at two unique sites simultaneously. The pairs of sites were selected to permit the nitrile symmetric stretching frequency of pCNF at each site to be spectrally resolved. The temperature dependent linear IR spectra of these sfGFP constructs with two pCNF UAAs incorporated will be presented, illustrating that the nitrile groups are in different local environments. Preliminary X‐ray crystal structures of these sfGFP constructs will be presented to probe if the UAAs resulted in a structural perturbation and to determine the distance between the nitrile groups of the two pCNF UAAs in each structure. These results will serve as the basis for and calibration of employing pCNF in future 2D IR spectroscopic studies to extract the distances between these two groups based upon coupling between them.