Probing Structural Implications of Unnatural Amino Acid Incorporation into Green Fluorescent Protein

Abstract

The ability to study local protein structure and dynamics has been greatly enhanced by the genetic incorporation of unnatural amino acids (UAAs) that contain spectroscopic reporters. An important characteristic of an effective spectroscopic reporter UAA is the ability to probe local protein environments in a relatively non-invasive manner. Here we have investigated the structural consequences of the genetic, site-specific incorporation of the spectroscopic reporter UAA 4-cyano-L-phenylalanine (pCNPhe) into distinct sites in superfolder green fluorescent protein (sfGFP) by X-ray crystallography. This UAA was selected since it can serve as both a vibrational and fluorescent reporter of local protein structure and dynamics. X-ray crystal structures of sfGFP constructs containing pCNPhe will be presented and the structural impacts of the incorporation of this UAA into sfGFP will be discussed.