Dissertation/Thesis Abstract

Studies of Membrane Protein Folding by Bimolecular Fluorescence Quenching
by Kozachenko, Ivan Andrew, M.S., University of California, San Diego, 2016, 164; 10159880
Abstract (Summary)

This project investigates changes in protein solvation during the folding reaction of Outer membrane protein A (OmpA) of Escherichia Coli, and correlates this dehydration process with shifts in local polarity. Changes in solvent accessibility were probed by fluorescence quenching experiments and Stern-Volmer analysis while polarity was monitored via emission maxima; site-specific insights were gained by probing single tryptophan OmpA mutants (W7, W15, W57, W102, W129, and W143). Fluorescence experiments were performed on OmpA denatured in 8.0 M urea, folded in small unilamellar vesicles (SUVs), adsorbed on SUVs, and aggregated in 0.5 M urea. Time resolved experiments were performed during the folding reaction. The Stern-Volmer quenching constant (KSV) and fluorescence maxima (λmax) showed a correlation where high values of KSV corresponded to red-shifted λ max. KSV and λmax values decayed from unfolded values of 8.6–10 M-1 and 351–354 nm to folded values of 0.6–2.0 M-1 and 325–338 nm. Double exponential fits to the KSV and λmax data showed fast (τ ~ 3–11 minutes) and slow components (τ ~ 26–49 minutes). The majority of λmax shift occurs in the fast step, while the majority of dehydration occurs in the slower step. The fast component was attributed to a transition of the trp residues of unfolded OmpA to a partially adsorbed state in the bilayer headgroup. A slower, dehydration event took place in which the protein fully inserted into the bilayer. These results complement previously proposed mechanisms of concerted folding, and provided insights into changes in solvation that accompanies formation of native structure.

Indexing (document details)
Advisor: Kim, Judy E.
Commitee: Komives, Elizabeth A., Tauber, Michael J.
School: University of California, San Diego
Department: Chemistry
School Location: United States -- California
Source: MAI 55/06M(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Physical chemistry, Biophysics
Keywords: Membrane protein, Protein, Protein folding
Publication Number: 10159880
ISBN: 9781369148084
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