Peptide toxin bioengineering of a voltage-gated potassium channel probe-utilizing azide chemistry in fluorescent bioconjugation

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2014-08
Authors
Halford, Zan Atherton
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[Honolulu] : [University of Hawaii at Manoa], [August 2014]
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Abstract
Scorpion venom toxins represent a large library of pharmacologically selective peptides that have been invaluable tools to providing the structural, functional, and pathological nature potassium ion (K+) channel subfamilies maintain in cells and tissues. Fluorescently engineered toxins are necessary for studying K+ channel isoforms in cells and tissues and have become a unique intersection between science and medicine as tools for advancing the pharmacology and treatment of channel implicated pathologies. Incorporating improved strategies of fluorescent bioconjugation to modify scorpion toxins is not only increasing the ease and efficiency of their syntheses but their applications as well by permitting living cell visualization of channel surface expression and function. Using solid-phase peptide synthesis (SPPS) techniques and a bioorthogonal conjugation to create a synthetic fluorescent Hongotoxin (Centruroides limbatus; HgTX) analog, a novel probe was created for the potassium voltage-gated channel, shaker-related subfamily, member 3 (Kv1.3). The linear SPPS of the 39 amino acid peptide and the ligation of an aliphatic azide constituent were optimized to demonstrate an ideal synthetic strategy for bioengineering larger peptide probe designs to contain functional unnatural modifications. Introducing the purified azido-HgTX analog to a fluorescent phosphine prosthetic has further demonstrated the chemoselectivity of these conjugation chemistries for use in the presence of biological molecules, holding the advantage over the classic methods, regarding their set of stringent reaction conditions during fluorescent probe construction.
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M.S. University of Hawaii at Manoa 2014.
Includes bibliographical references.
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isoforms
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Theses for the degree of Master of Science (University of Hawaii at Manoa). Molecular Biosciences and Bioengineering.
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