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Principal Investigator: Scott H Brewer
Organization: FRANKLIN AND MARSHALL COLLEGE
Fiscal Year: 2024
Award: $423,133
Funding agency: National Institute of General Medical Sciences
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.
Terms: <2-dimensional><AMP Kinase><ATP-AMP Phosphotransferase><ATP-AMP Transphosphorylase><ATP-protein phosphotransferase><Adenylokinase><Austria><Azides><Buffers><CRISPR interference><CRISPR-dCas9-mediated repression><CRISPR/dCas9 interference><CRISPR/dCas9-mediated transcriptional inhibition><CRISPRi><Chemicals><Ciliary Dyskinesia><Ciliary Motility Disorders><Clustered Regularly Interspaced Short Palindromic Repeats interference><Collaborations><Complex><Coupled><Coupling><Crystallographies><Crystallography><Data><Deoxyuridine><Development><Effectiveness><Electrostatics><Environment><FRET><Fluorescence Resonance Energy Transfer><Frequencies><Future><Förster Resonance Energy Transfer><Genetic Alteration><Genetic Change><Genetic defect><Goals><Green Fluorescent Proteins><H-bond><Health><Hemolytic Anemia><Hydration><Hydration status><Hydrogen Bonding><Immotile Cilia Syndrome><Individual><Isotope Labeling><Kinase Family Gene><Kinetics><Measurement><Measures><Methodology><Methods><Molecular><Mutation><Myokinase><Nevada><Nitriles><Non-Polyadenylated RNA><Nucleic Acids><Nucleosides><Nucleotides><Pathway interactions><Phenylalanine><Play><Protein Kinase><Proteins><RNA><RNA Gene Products><RNA Interference Therapy><RNA interference therapeutics><RNA interference-based therapy><RNA vaccine><RNA-based vaccine><RNAi therapeutics><RNAi therapy><RNAi-based therapeutics><RNAi-based therapy><Reporter><Research><Ribonucleic Acid><Roentgen Rays><Role><Single Crystal Diffraction><Site><Solvents><Specificity><Stretching><Structure><Students><System><Temperature><Universities><Variant><Variation><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><adenylate kinase><college><collegiate><developmental><dimer><experiment><experimental research><experimental study><experiments><genome mutation><glycogen synthase a kinase><hydroxyalkyl protein kinase><infrared spectroscopy><mRNA vaccine><mRNA-based vaccine><monomer><non-natural amino acids><non-proteinogenic amino acids><nonproteinogenic amino acids><nucleic acid structure><pathway><phosphorylase b kinase kinase><repressing CRISPR-dCas9 system><reticular dysgenesis><simulation><social role><temporal measurement><temporal resolution><time measurement><two-dimensional><undergrad><undergraduate><undergraduate research><undergraduate student><unnatural amino acids>