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dc.contributor.authorRivera-Torres, Ivan O.
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 65-07, Section: B, page: 3451.;Advisors: Mark E. Girvin.
dc.description.abstractBacillus pseudofirmus OF4 accomplishes optimal aerobic growth at pHout 10.5, while the pHin remains at 8.3. The transmembrane pH difference (DeltapH), diminishes the protonmotive force (Deltap), while there is an insufficient rise in the transmembrane electrical potential (DeltaPsi). However oxidative phosphorylation measured by a phosphorylation potential (DeltaGp), increases. The imposition of artificial diffusion potentials fail to energize ATP synthesis above pHout of 9.3.;Protonation of OF4 subunit c, could occur via a localized circuit active at high external pH, from protons directly transferred into the F1Fo ATP syntheses. Amino acid substitutions in OF4 c (G17, A20, A22, K26, T33 and P51) were assumed to confer structural adaptations relevant to alkaliphilicity. Mutants where these residues are replaced by those in the neutralophile consensus assemble an active ATPase/ATP synthase, although their growth capability in non-fermentative media and ATP synthetic rates is strongly diminished (Wang et al., 2004).;We have studied the OF4 c at pH 7.8 via NMR spectroscopy, to provide an insight into the structural role of these residues. The expression and purification of OF4 c was optimized. Good sample conditions were achieved in 4:4:1 CHCl3/CH3OH/H2O, supporting structural comparisons with the E. coli c structure. 3D NMR experiments were collected to fully assign the NMR resonances. A pH titration study enabled us to estimate a pKa of 7.74 for E54, and suggested that a single major conformational transition occurs above this pKa. OF4 c folds as a helix-loop-helix, with interhelical contacts demonstrated by paramagnetic relaxation effects.;The structural contribution of the alkaliphilic residues, was evaluated in models for OF4 c, generated by simulated annealing and torsion angle dynamics, using chemical shift assignments and NMR-derived structural restraints. The best models were superimposed onto the structure of E. coli c, for structural comparisons. The alkaliphilic residues assume various roles, such as the preservation of interhelical distances, facilitation of packing interactions between helices and/or monomers, establishment of physical contacts for effective rotation within Fo and F 1, and modulation of the local structure and/or flexibility affecting Glu54, perhaps enhancing proton transfer. A model of OF4 subunit a is also presented.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleStructural studies of the proton-translocating subunit of an alkaliphilic F(1)F(o) ATP synthase

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