Browsing by Author "Kennan, Alan J., advisor"

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Open Access
Heterotrimeric coiled-coils as viral fusion protein mimics
(Colorado State University. Libraries, 2010) Johnson, Dana E., author; Kennan, Alan J., advisor; Finke, Richard G., committee member; Peersen, Olve B., committee member; Rovis, Tomislav, committee member; Shores, Matthew P., committee member
The a-helical coiled-coil, formed by the association and supercoiling of two or more a-helices, is a ubiquitous protein structure that mediates a wide range of biological activity. It is characterized by a heptad repeat of amino acids that serve to form both the hydrophobic core and electrostatic interfaces of the coiled-coil. Previous work in our lab showed the viability of designing a self-assembling heterotrimeric coiled-coil by sole manipulation of the hydrophobic core. This technique utilized a steric matching approach whereby one large side chain packed against two small ones. An added benefit to this type of control is that it allowed the freedom to explore additional interactions specific to the electrostatic interface. Many enveloped viruses, including HIV-1, incorporate trimeric coiled-coils in their fusion proteins, and, consequently, are involved in the pathway to infection. Through interactions at the electrostatic interfaces of the coiled-coils, these fusion proteins form a six-helix bundle called a trimer-of-hairpins. The formation of this structure is a precursor to membrane fusion of the viral and host cells, and, as a result, it has become a therapeutic target. The steric matching technique developed in our lab allows us to graft key contacts from the native 111 sequences onto a stable, heterotrimeric system and construct a mimic of the trimer-of-hairpins, as was done with HIV-I previously in our lab. The work that follows shows that a viable mimic of the Human Respiratory Syncytial Virus is also possible. A stable, self-assembling mimic was designed, synthesized and validated through various spectroscopic methods. Additionally, a mutant study was conducted to further refine our knowledge of the importance of the residues thought to be key to the formation of the trimer-of-hairpins. Other work was performed extending the process to the Human T-cell Leukemia Virus, bringing the possibility of a complete and stable mimic ever closer.
Open Access
Investigating biosynthetic pathways of the Aspergillus genus through biomimetic total synthesis of secondary metabolites
(Colorado State University. Libraries, 2022) Benson, Brooke, author; Williams, Robert M., advisor; Kennan, Alan J., advisor; Paton, Robert, committee member; Crans, Debbie, committee member; Crick, Dean, committee member
The prenylated indole alkaloids are a class of secondary metabolites containing a unique bicyclo[2.2.2]diazaoctane core and a wide range of biological activity. This complex structure has prompted extensive investigation into the biochemical synthesis of these compounds. Currently, three disparate biochemical strategies are known to be used by producing fungi to construct the bicyclic core: (1) NADPH-dependent bifunctional reductase/Diels-Alderase-mediation in formation of the monooxopiperazines; (2) brevianamide assembly through cofactor-independent pinacolase resulting in spontaneous intramolecular Diels-Alder (IMDA) generation of the bicyclo[2.2.2]diazaoctane core; (3) Diels-Alderase mediated enantiodivergent generation of the dioxopiperazines via cytochrome P450 oxidation to achiral azadienes and successive enzyme-mediated stereoselective IMDA reaction. This work aimed to employ biomimetic total synthesis to aid in elucidation of the biosynthetic pathways in the Aspergillus genus, which utilizes the third strategy. This author reports the first total syntheses of 6-epi-Notoamides T10-12 and Notoamide T2, as well as an improved total synthesis of 6-epi-Notoamide T. Also reported are synthetic efforts towards 6-epi-Notoamide T9, Notoamide TI, and Citrinalin C.
Open Access
Trimeric coiled-coils as viral fusion protein mimics
(Colorado State University. Libraries, 2008) Travisano, Philip, III, author; Kennan, Alan J., advisor
α-Helical coiled-coils, a protein structural motif formed by supercoiling of two or more component polypeptide strands, are ubiquitous mediators of biological structure and function. Their characteristic primary heptad repeat, denoted abcdefg, makes these complexes attractive scaffolds for studying self-assembly and molecular recognition. Assembly of these structures is driven by the hydrophobic effect in which the hydrophobic sidechains associated with positions a and d are specifically packed together. Recently we have described methods for controlling the assembly of 1:1:1 heterotrimeric coiled-coils using only interior hydrophobic core residues. These core residues assemble according to steric matching, one large sidechain packs against two small sidechains. In the following text we have explored new sidechain parings. This steric matching strategy affords maximal sequence flexibility in the patterning of exterior surface residues, which we have exploited to create mimics of therapeutically significant protein-protein interfaces. The Human Immunodeficiency Virus (HIV) envelope protein gp41 facilitates infection by promoting fusion of cellular and viral membranes. At the heart of its function is formation of a trimer-of-hairpins structure in which a C-terminal ligand peptide binds to an N-terminal coiled-coil surface. This interaction is reminiscent of those in numerous other viral systems, including visna, the sheep analog of HIV. The design of protein mimics for viral systems by installation of key contact residues onto heterotrimer coiled-coils will be further discussed. The following text will highlight the structural verification of these mimics through various spectroscopic techniques. Also the validation of these mimics will be tested by exposure to known viral inhibitors. The work included in this text builds on previous research conducted in our laboratory, but it provides new avenues for future projects to explore the detailed interactions within the viral fusion mimics. This will hopefully lead to a better understanding of the viruses being studied as well as the underlying molecular interactions taking place.