Dissertation
Association 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-chains
Abstract
To 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.
Citation
@phdthesis{doi:10.17170/kobra-202010231991,
author={Gholami, Sara},
title={Association 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-chains},
school={Kassel, Universität Kassel, Fachbereich Mathematik und Naturwissenschaften, Institut für Biologie},
year={2020}
}
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4000 Association 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-chains / Gholami, Sara
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<resource xsi:schemaLocation="http://datacite.org/schema/kernel-2.2 http://schema.datacite.org/meta/kernel-2.2/metadata.xsd"> 2020-10-26T11:18:07Z 2020-10-26T11:18:07Z 2020 doi:10.17170/kobra-202010231991 http://hdl.handle.net/123456789/11894 eng Urheberrechtlich geschützt https://rightsstatements.org/page/InC/1.0/ membrane protein protein folding hvdac1 570 Association 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-chains Dissertation To 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. open access Gholami, Sara 2020-10-02 vi, 147 Seiten Kassel, Universität Kassel, Fachbereich Mathematik und Naturwissenschaften, Institut für Biologie Kleinschmidt, Jörg Helmut (Prof. Dr.) Herberg, Friedrich Wilhelm (Prof. Dr.) Membranproteine Proteinfaltung Voltage-Dependent Anion Channels publishedVersion false </resource>
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