PROJECT SUMMARY The long-term objective of this research is to use vibrational reporters and infrared (IR) spectroscopy to study protein and nucleic acid structure and dynamics. A full understanding of these requires a sensitive, site-specific, and relatively non-invasive methodology with sufficient spatial and temporal resolution. Nitrile (CN) and azide (N3) vibrational reporters satisfy these requirements when coupled with IR spectroscopy. The vibrational reporter unnatural amino acid (UAA) 4-cyano-L-phenylalanine (CNPhe) will be site-specifically genetically incorporated, individually into numerous sites in adenylate kinase (AK). The local environments of the nitrile group of CNPhe will then be assessed upon thermal unfolding of the protein with temperature-dependent linear IR spectroscopy. The molecular interpretation of these spectroscopic results will be aided by X-ray crystallography in collaboration with Christine Phillips- Piro (F&M). The aggregation mechanism and kinetics of AK will also be explored with CNPhe and IR spectroscopy. The ability to potentially enhance the effectiveness of the nitrile reporter of CNPhe and 5- cyano-2'-deoxyuridine (CNdU) will be explored by correlating the temperature-dependent nitrile symmetric stretch frequency with either the temperature-dependent 13C or 15N NMR chemical shifts of isotopically labeled variants of these molecules. The goal will be to gain a better understanding of the hydration and electrostatic effects impacting the nitrile symmetric stretching frequency. This method will then be extended to superfolder green fluorescent protein (sfGFP). The further development of a method to extract the geometric orientation between two vibrational reporters using 2D IR spectroscopy in biomolecules will also be pursued. In collaboration with Matthew Tucker (University of Nevada, Reno), the anharmonic coupling between the vibrational reporter pairs, measured by 2D IR spectroscopy, will be correlated to the distance and angle between these reporters in several systems including: a nucleoside based hydrogen-bonded dimer system, a RNA tetraloop hairpin (prepared in collaboration with Ronald Micura, University of Innsbruck, Austria), sfGFP, and adenylate kinase (AK) constructs, where each system contains either two nitrile reporters or one nitrile and one azide reporter. Franklin & Marshall College undergraduate students will play an integral role in this biomedical health-related research.