Date of Award
Bachelor of Science
Jason P. Seeley
John T. Welch
The preparation and characterization of β-sheet forming polypeptides for use in light harvesting applications is described. The Higashiya genetic cassette strategy (HGCS) was employed in order to make DNA libraries of repetitive DNA sequences. These DNA libraries contained gene cassettes coding for proteins designed to fold into the desired secondary motif. The conditions required for peptide expression, folding and formation of micron length fibrils was determined for the purpose of photophysical characterization, such as fluorescence lifetimes and acceptor dye quantum yields. Of special interest was the investigation of an “antenna effect” under FRET illumination, the intensity of acceptor dye emission was examined as a consequence of increasing peptide length. When β-sheet forming polypeptides bearing tryptophan residues along one edge were irradiated at 280 nm, the tryptophan absorbance maximum, the fluorescence intensity of the C-terminal acceptor chromophore, N-(7-dimethylamino-4-methyl) coumarin (DACM), increased proportionally with an increasing number of donor tryptophans. The fluorescence intensity increased even as the length of the β-sheet edge approached 256 Å, well beyond the Förster radius for the tryptophan-acceptor chromophore pair. Folding of the polypeptides was examined as well using Circular Dichroism, and fibrillation was examined using ThT kinetic studies. Beta sheet formation was detected via Circular Dichroism, and fibrillation was confirmed by the ThT kinetics studies. The plots of CPS vs. time showed an increase in florescence which was indicated by the positive slope of the plot, this positive slope shows that fibrillation is occurring.
Eagleton, Aileen M., "Genetically Engineered Peptides for Light Harvesting and Folding Kinetics Studies" (2018). Chemistry. 11.