Skip to Main Content
 

Global Search Box

 
 
 
 

Files

ETD Abstract Container

Abstract Header

Peptide Tertiary Structure and Fusion Peptide

Torres, Oscar Buena

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Chemistry.
In this work synthetic peptides were used to study peptide tertiary structure nucleation and to probe the structural determinants of membrane activity. In the first study, we “crippled” 21-residue sequences derived from the GCN4 leucine zipper by positioning glycine residues in the c and e helix positions of the central heptad, and by leaving the hydrophobic core residues in the a and d positions intact as isoleucine and leucine, respectively. Crosslinking residues (X = Histidine or Azido alanine) were placed in the b and f (i and i + 4) positions to yield the crippled histidine and crippled azidoalanine peptides. Restoration of the secondary and tertiary structures of the crippled histidine peptide was effected by metal complexation. Indeed, the circular dichroism (CD) spectrum of a crippled histidine sequence exhibited a saturable increase in helicity upon treatment with NiCl2, which can be reversed with EDTA. While the free peptide was a completely unfolded monomer, the resulting nickel-complexed peptide melted cooperatively with a Tm of 46 °C, and was found by analytical ultracentrifugation (AUC) to be dimeric. Helix turn stabilization and peptide tertiary structure nucleation of the crippled azidoalanine peptide were probed by double “click” cyloaddition. In this strategy, azidoalanine residues (i and i + 4) were linked by bis-alkynes: meta-diethynylbenzoic acid, ortho-diethynylbenzoic acid, dipropargylated glycine, and hexa-1,5-diyne. Ring-closed adduct of meta-diethynylbenzoic acid was insoluble in aqueous solvent. On the contrary, peptide cycloadducts derived from ortho-diethynylbenzoic acid and dipropargylated glycine were soluble in aqueous medium. The cycloadduct of ortho-diethynylbenzoic acid exhibited a random coil signature with and without NiCl2, while the cycloadduct of dipropargylated glycine revealed an increase in helicity upon treatment with NiCl2. AUC measurements for dipropropargylated glycine adduct indicated a monomer–dimer equilibrium, which suggests that metal complexation did not completely restore dimerization. Commercially available hexa-1,5-diyne, which induced the most significant change in helicity upon double “click” cycloaddition, provided sufficient efficiency to restore dimerization to the folding-crippled azidoalanine. Like the nickel complex of crippled histidine, the hexa-1,5-diyne adduct peptide melted cooperatively (Tm = 40 °C) but dimerized independently of nickel concentration. We confirmed that complete ring closure and not the installation of triazoles restored the observed tertiary structures of hexa-1,5-diyne cycloadduct through a Staudinger reduction and a MS-MS peptide fragmentation pattern. Furthermore, cycloadducts of crippled azidoalanine and crippled azidoserine (i.e. one azidoalanine residue was replaced with serine) with propargyl alcohol remained unstructured under all conditions. In the second study, we synthesized a small library of 38 variants of the 23-residue fusion peptide domain found at the N-terminus of gp-41 glycoprotein of HIV. This hydrophobic, glycine-rich sequence is critical for viral infectivity and is thought to be a central component in the membrane fusion of viral envelope with the host membrane. There has been extensive discussion in the literature regarding the origin of fusogenicity in this viral fusion sequence. Our library of fusion peptide variants was designed to address the biophysical importance of secondary structure, peptide flexibility, glycine content and placement. We assayed each peptide for its ability to induce lipid-mixing (FRET dilution) and membrane-permeabilization in synthetic vesicles (Calcein leakage). We find that the viral fusion peptide required may be greatly simplified while retaining fusogenic function and minimizing membrane-permeablizing function; to the best of our knowledge, this is the first attempt to optimize fusogenic function of the HIV fusion peptide through sequence variation. Our data show that many flexible, linear, minimally hydrophobic peptides may achieve the biophysical function of fusion; glycine does not appear to be essential. These findings will be useful in the design of synthetic fusogens for cellular delivery.
Dennis Bong, PhD (Advisor)
Jovica Badjic, PhD (Committee Member)
Karin Musier-Forsyth, PhD (Committee Member)
229 p.

Recommended Citations

Citations

  • Torres, O. B. (2011). Peptide Tertiary Structure and Fusion Peptide [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300819582

    APA Style (7th edition)

  • Torres, Oscar. Peptide Tertiary Structure and Fusion Peptide. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1300819582.

    MLA Style (8th edition)

  • Torres, Oscar. "Peptide Tertiary Structure and Fusion Peptide." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1300819582

    Chicago Manual of Style (17th edition)