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Peptide Tertiary Structure and Fusion Peptide

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Peptide Tertiary Structure and Fusion Peptide Peptide Tertiary Structure and Fusion Peptide Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Oscar B. Torres, B.S. Graduate Program in Chemistry The Ohio State University 2011 Dissertation Committee: Dr. Dennis Bong, Advisor Dr. Jovica D. Badjic Dr. Karin Musier-Forsyth Copyright by Oscar B. Torres 2011 Abstract 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 ii 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 iii 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. iv Dedication To my mother Leonora B. Torres for her unconditional love And To my family. v Acknowledgements This work would not have been possible without the assistance and encouragement of my colleagues in the Biological Chemistry Division. My journey in graduate school has enriched my growth as an independent researcher and educator. I would like to take this opportunity to pay tribute to those who have helped me achieve this academic pursuit. First and foremost, I would like to thank my mentor, Professor Dennis Bong. It has been an honor to be a part of his research team. I am very grateful for his enumerable contributions that made this work come to fruition. Professor Bong has given constructive criticism that helped me evolve as an effective researcher. Besides scientific knowledge, Professor Bong helped me to mature as an individual with a strong work ethic and an unprecedented perseverance. As he often reminds me: “Try and try until the system works. Don’t give up unless you have completely destroyed the sample”. I am indebted to him for my research accomplishments. I am grateful to have Professor Sean Taylor as my former adviser. He gave me the opportunity to improve my synthesis skills. I would like to thank my dissertation committee, Professor Jovica D. Badjic and Professor Karin Musier-Forsyth for offering invaluable critiques and suggestions. I am grateful to my oral examination committee, vi Professor Thomas Magliery, Professor Ross Dalbey and Professor Karin Musier-Forsyth for believing in my capability to finish the chemistry doctorate program. At Bong group, I was able to gain experience in different facets of scientific inquiry, such as small and polymer syntheses, cell-based assays, and biophysical techniques. I appreciate Dr. Saibal Bandyophadthay, Dr. Soumitra Chatterje and Dr. Mingming Ma for giving suggestions in organic synthesis and Dr. Yun Gong for helping me establish good habits in the cell culture lab. I would like to extend my appreciation to the present and past members of Bong group who provided camaraderie and encouragement whenever I was frustrated with my experiments. I especially acknowledge Yingying Zeng, who always provides a helping hand whenever I was fixing HPLC, peptide synthesizer, and lyophilizer. For doing exceptional preliminary studies on nickel-histidine complex, I would like to thank Matt Bernardina and our collaborators Prof. Krishna Kumar and Deniz Yuksel for doing ultracentrifugation experiment, and also Liwen Zhang for the MS-MS experiment. I would like to thank the Musier-Forsyth group for letting me use their gel apparatus during my preliminary experiments; Magliery group for sharing their M5 plate reader and the JASCO CD apparatus; and Paulatis group for allowing me to use their Biosafety hood for cell-based assay. My time in Columbus has been prolific due to friends that have become part of my life. I would like to thank Jomarie Bacusmo for being a colleague and a friend. She always uplifts my spirit whenever I was down. Your critique on improving my presentation meant so much to me. Beyond graduate school, Erik Lauritzen has been a vii good housemate and a good friend. Erik assisted me to improve my diction and has provided my deep understanding of the American culture. His daily critique helped me to be an effective TA in Chemistry 251. The Lauritzen family was my home away from home, and has been the source of courage whenever I felt nostalgic for my family. I would like to thank the Fulbright Program for making my graduate studies in the United States feasible. I could never have embarked on this journey without their generosity. The Fulbright Program has given me the opportunity to attend gateway orientations and conferences where many countries were represented. Indeed, the program has given me experience beyond the lab bench and has nourished my growth as a leader. Finally, I would like to thank my family for their unwavering support and constant prayers. My mother, Leonora B. Torres, has instilled the importance of education and empathy. My sincere apologies to those whom I have not personally mentioned, but have also helped me in various ways towards the realization of my ambition. viii Vita 1997 .................................................................................. Camarines Norte State College 2001...................... B.S. Chemistry (cum laude), University of the Philippines Los Banos 2001-2004 ..................................... Instructor II , University of the Philippines Los Banos 2004-2011 ......... Fulbright Scholar, Department of Chemistry, The Ohio State University 2004-2011 ...... Graduate Associate, Department of Chemistry, The Ohio State University Publications Oscar B. Torres, Deniz Yuksel, Matt Bernardina, Krishna Kumar, and Dennis Bong. Peptide tertiary structure nucleation by sidechain crosslinking with metal complexation and double "click" cycloaddition. ChemBioChem
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