| This dissertation describes the use of highly fluorinated analogues of proteinogenic β3-amino acid side chains to enhance the properties of β3-peptide bundles. β 3-Peptides are non-natural polyamides that differ from naturally occurring α-peptides by the addition of a single methylene unit in the backbone of each constituent β 3-amino acid. They are able to fold into stable secondary, tertiary, and quaternary structures depending on their primary sequences and can be functionalized to interact with natural biomolecules. Due to their structural and functional propensities, along with their proteolytic stability, there is much interest in the use of β3-peptides as proteomimetics. Two possible avenues for making β3-peptide bundles more protein-like are the incorporation of a unique cellular localization and the diversification of the hydrophobic core, both of which are described herein.;In order to direct the assembly of β3-peptide helical bundles in membranes, the highly fluorinated amino acid β3-6,6,6,6',6',6'-hexafluoro-homoleucine was incorporated into hydrophobic β3-peptide 14-helices because the fluorous effect has been shown to induce oligomerization of α-helical coiled coil peptides in lipid bilayers. First, the fluorous residue was introduced to the cores of the aqueous octameric β3-bundles, Zwit-1F and Zwit-EYYK, to ensure the substitution was tolerated and to test for the fluoro-stabilization effect. This resulted in the formation of a stable octameric β 3-bundle with a single, central fluorous core. During the biophysical characterization of this peptide, interesting new properties of the fluorous effect were discovered, including elevated cold denaturation temperatures of the peptide and the enhanced fluorescent response of a small organic dye.;Subsequently, the charged and polar residues on the aqueous bundle exterior were replaced with functionally analogous hydrophobic residues to promote membrane solubility of the peptides, and characterization was carried out in micelles and vesicles. Although these hydrophobic and fluorous peptides did not display self-association in the membrane environments, there was spectroscopic, chromatographic, and fluorescent evidence that these molecules were the first β 3-peptides to form stable transmembrane helices.;Regarding the diversification of β3-bundle core sequences, the protein modeling software package Rosetta was used to predict substitutions to the β3-homoleucine core of Acid-1Y that would result in stabilized octameric β3-bundles. The best result was for a double β3-homophenylalanine substitution, which assembled into an octamer as predicted, but with diminished stability. Rational replacement of one of the two β3-hPhe residues with β 3-pentafluoro-homophenylalanine led to an electrostatically favorable face-to-face interaction within the core due to the reversed quadrupoles of these two aromatic side chains. This was evidenced by improved β3-bundle stability and greater organization within the hydrophobic core. These experiments represented the first application of Rosetta modeling to non-natural folding oligomers and resulted in the two β3-peptide bundles with the most diverse core sequences. |
