Date of Award
1-1-2025
Document Type
Thesis
Degree Name
M.S. in Biological Science
First Advisor
Mika Jekabsons
Second Advisor
Bradley Jones
Third Advisor
Joshua Bloomekatz
School
University of Mississippi
Relational Format
dissertation/thesis
Abstract
This study investigates Bax binding to voltage-dependent anion-selective channels (VDAC1 and VDAC2) N-terminal 26-mer peptides, which have strong sequence homology to BH3 domains, to assess binding stoichiometries, affinities, residues involved in binding, and the potential role of peptide secondary structure in this process. Peptide helical content was assessed without or with Fos12 detergent using circular dichroism spectroscopy. Fluorescent-tagged peptides were used to assess binding affinities and stoichiometries to recombinant Bax, after correction for non-specific binding using bovine serum albumin. Affinities for VDAC1 and VDAC2 were three- to six-fold, respectively, lower than for the Bim BH3 domain, but all three peptides bound with a stoichiometry of one site per monomer. All three wild type peptides displayed ~67 % helical content with Fos12 present, whereas secondary structure was moderately to substantially reduced when mutations were introduced. Reduction in helical content strongly correlated with decreased affinity for a subset of peptides, whereas others exhibited affinities that were either strictly due to the introduced sequence modification, or a combination of the sequence modification and loss of secondary structure. Notably, the conserved aspartate (D16 or D27 for human VDACs 1 & 2, respectively) was important for Bax binding. Some double hydrophobic residue VDAC mutants bound with a stoichiometry of two sites per monomer, supporting previous work indicating two distinct BH3 domain sites on Bax. The results are consistent with the possibility that VDACs 1 & 2 interact with Bax through their N-terminal BH3-like domains.
Recommended Citation
Greico, Mackense Kathleen, "Voltage-Dependent Anion-Selective Channels 1 and 2 Wild Type and Mutant BH3-Like Domain Peptides: Analysis of Secondary Structure and Binding Stoichiometry to Recombinant Bax" (2025). Electronic Theses and Dissertations. 3287.
https://egrove.olemiss.edu/etd/3287