Author ORCID Identifier
Document Type
Thesis
Date of Award
Fall 12-10-2024
Keywords
HhC, cholesterol, epi-coprostanol, coprostanol, cholestanol, FRET, error prone PCR, NAMD
Degree Name
Biochemistry (BS)
Department
CHEMISTRY
First Advisor
Dr. Brian P Callahan
Series
Science and Mathematics
Subject Heading(s)
Hedgehog proteins--Structure-activity relationships
Abstract
Sonic hedgehog protein is vital for embryonic development while also being linked to multiple cancers and holoprosencephaly. The native reaction of hedgehog is a sterolysis reaction whereby cholesterol binds to the HhC catalytic domain and attacks the HhN signaling domain, resulting in a covalent HhN-cholesterol signaling product. The structure of the sterol recognition region (SRR) of Sonic Hedgehog, where cholesterol binds, is currently unknown. In this thesis, I used directed evolution to select a triple mutant of Hedgehog’s catalytic HhC domain, (D46H, Q150H, A196T), which is over 5000 times more catalytically efficient at reacting with epicoprostanol (ECP), a non-native substrate and an inhibitor of wild-type hedgehog protein. A sterol screen of the mutant revealed that it also accepts lithocholic acid (LCA) as a substrate, which is promising for future bioconjugation experiments. Investigations into the role of each mutation reveal the necessity of A196T in accepting LCA’s carboxyl tail, the significance that Q150H has on hydrolysis, as well as the necessary and synergistic effects of D46H. Computational models of HhC give some insight into the structural role of the mutations, but the largely disordered loop region of the SRR leaves more questions unanswered about its structure.
Recommended Citation
Kuke, Ted, "Directed Evolution of Hedgehog Protein Autoprocessing Activity: Combined Experimental & Computational Approach" (2024). Undergraduate Honors Theses. 49.
https://orb.binghamton.edu/undergrad_honors_theses/49
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