Probing Conformational Changes of E. coli Adenylate Kinase Using a Vibrational Reporter

Abstract

Unnatural amino acids (UAAs) consisting of a vibrational reporter such as a nitrile, azide, or nitro group have the potential to serve as effective, site-specific probes of local protein environments. Specifically the UAA 4-cyano-L-phenylalanine (pCNF) contains a nitrile vibrational reporter that is an effective probe of local environment due to the position and sensitivity of the nitrile symmetric stretch to local environment, in addition to the relative small size of the nitrile group. Additionally, this UAA can be site-specifically incorporated into proteins using the amber codon suppression method. Here, this vibrational reporter UAA was genetically incorporated at multiple distinct sites in the enzyme adenylate kinase (AK), which catalyzes the conversion of two adenylate diphosphate (ADP) molecules to one adenosine monophosphate (AMP) molecule and one adenosine triphosphate (ATP) molecule. Multiple sites were selected to represent a myriad of local solvation states some of which are predicted to change upon nucleotide binding based upon the X-ray crystal structures of apo and nucleotide bound AK. The local solvation states will be further characterized through the use of temperature-dependent infrared spectroscopy (temp-dep-IR) under various nucleotide binding conditions.