Probing Local Protein Environments with a Vibrational Reporter Unnatural Amino Acid

Abstract

Vibrational reporter unnatural amino acids have the potential to serve as sensitive, site-specific probes of local protein environments. Here the vibrational reporter unnatural amino acid 4-cyano-L-phenylalanine (pCNF) was utilized to probe multiple distinct sites in the Heme Nitric Oxide and/or Oxygen (H-NOX) binding domain from Caldanaerobacter subterraneus. pCNF is an effective reporter of local environment due to the position and sensitivity of the nitrile stretch frequency. pCNF was genetically incorporated into H-NOX into three distinct sites using the amber codon suppression methodology. The sites were selected to represent a myriad of local protein environments. The frequency of the nitrile symmetric stretch of pCNF and the temperature dependence of this frequency was utilized to assess the solvation state of the nitrile group in each of these sites in the protein. This analysis was aided by measuring the frequency and temperature dependence of the nitrile stretch vibration of pCNF in a variety of solvents to mimic different protein environments. These results probing the local environments in H-NOX will be presented.