Show simple item record
dc.rightsUrheberrechtlich geschützt
dc.subjectmembrane proteineng
dc.subjectprotein foldingeng
dc.titleAssociation of neighboring β-Strands to form the β-barrel structure of the voltage-dependent anion channel, human isoform 1 (hVDAC1) precedes membrane insertion and is largely driven by polar interactions between basic and acidic amino acid side-chainseng
dcterms.abstractTo date, all biophysical folding studies on β-barrel membrane proteins from outer membranes (OMPs) indicated a coupled mechanism of folding and membrane insertion. Most of these studies were performed with smaller OMPs from bacteria. Here we have investigated folding and insertion of a mitochondrial OMP, the voltage-dependent anion-selective channel (VDAC), human isoform 1 (hVDAC1). To examine whether folding and insertion are coupled for hVDAC1, the association of neighboring β-strands of the 19-stranded β-barrel of hVDAC1 was studied in the absence and in the presence of lipid bilayers. The formation of antiparallel β-strands and of the parallel β-strands 1 and 19 that close β-barrel were examined by site-directed fluorescence quenching. Based on a gene encoding a mutant of hVDAC1, in which the four native tryptophans (W) were replaced by phenylalanine and the two native cysteines (C) were replaced by alanine, several fluorescent single-C, single-W- hVDAC1 mutants (XnC-ZmW-FhVDAC1) were designed, expressed and isolated. The C and the W replaced residues X and Z, that were selected for their structural proximity at positions n and m in two adjacent β-strands of the β-barrel. The C was labeled with a nitroxide spin label, which is a short-range quencher of fluorescence. The association of the neighboring β-strands upon folding of hVDAC1 was then investigated by intramolecular quenching of the tryptophan (W) -fluorescence by the nitroxide-label covalently linked to the SH-group of the C. Fluorescence spectroscopy demonstrated that contacts between neighboring β-strands are not observed for the urea-denatured mutants of hVDAC1. Regardless of its folding state and environment (unfolded in 8 M urea, in aqueous buffer after urea dilution, in detergent micelles or lipid bilayers), contacts were also never observed for another mutant used as a negative control, I138C-L275W-FhVDAC1, in which C and W are in β-strands 9 and 19 and thus neither in neighbor strands nor in structural proximity. In contrast, all specifically designed mutants of hVDAC1 that contain W and C in proximity in the high-resolution structure, formed an aqueous intermediate with a native-like β-barrel structure as indicated by intramolecular site-directed fluorescence quenching and CD spectroscopy. The fluorescence quenching in these aqueous intermediates formed after urea-dilution was very similar to the fluorescence quenching of the mutants folding and insertion into lipid bilayers, indicating that in contrast to previous observations for bacterial OMPs, the β-barrel of hVDAC1 is largely formed already in the aqueous folding intermediate in the absence of lipid or detergent.eng
dcterms.accessRightsopen access
dcterms.creatorGholami, Sara
dcterms.extentvi, 147 Seiten
dc.contributor.corporatenameKassel, Universität Kassel, Fachbereich Mathematik und Naturwissenschaften, Institut für Biologieger
dc.contributor.refereeKleinschmidt, Jörg Helmut (Prof. Dr.)
dc.contributor.refereeHerberg, Friedrich Wilhelm (Prof. Dr.)
dc.subject.swdVoltage-Dependent Anion Channelsger

Files in this item


This item appears in the following Collection(s)

Show simple item record