Aufsatz
The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers
Abstract
Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or β-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual β-strands fold. Here, the folding and insertion of the β-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (β9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand β9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the β-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand β9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins.
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Gefördert durch den Publikationsfonds der Universität KasselCitation
@article{doi:10.17170/kobra-202303157642,
author={Herwig, Sascha and Kleinschmidt, Jörg Helmut},
title={The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers},
journal={Membranes},
year={2023}
}
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3000 Herwig, Sascha
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4000 The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers / Herwig, Sascha
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5550 {{Membranproteine}}
5550 {{Transmembranproteine}}
5550 {{Proteinfaltung}}
5550 {{Lipide}}
5550 {{Doppelschicht}}
5550 {{Protein OmpA}}
5550 {{Fluoreszenzspektroskopie}}
7136 ##0##http://hdl.handle.net/123456789/14527
<resource xsi:schemaLocation="http://datacite.org/schema/kernel-2.2 http://schema.datacite.org/meta/kernel-2.2/metadata.xsd"> 2023-03-24T15:03:17Z 2023-03-24T15:03:17Z 2023-02-19 doi:10.17170/kobra-202303157642 http://hdl.handle.net/123456789/14527 Gefördert durch den Publikationsfonds der Universität Kassel eng Namensnennung 4.0 International http://creativecommons.org/licenses/by/4.0/ outer membrane protein protein folding lipid bilayer β-barrel BamA OmpA cysteine scanning mutagenesis site-directed fluorescence spectroscopy BAM complex 570 The Formation of β-Strand Nine (β9) in the Folding and Insertion of BamA from an Unfolded Form into Lipid Bilayers Aufsatz Transmembrane proteins span lipid bilayer membranes and serve essential functions in all living cells. Membrane-inserted domains are of either α-helical or β-barrel structure. Despite their biological importance, the biophysical mechanisms of the folding and insertion of proteins into membranes are not well understood. While the relative composition of the secondary structure has been examined by circular dichroism spectroscopy in folding studies for several outer membrane proteins, it is currently not known how individual β-strands fold. Here, the folding and insertion of the β-barrel assembly machinery protein A (BamA) from the outer membrane of Escherichia coli into lipid bilayers were investigated, and the formation of strand nine (β9) of BamA was examined. Eight single-cysteine mutants of BamA were overexpressed and isolated in unfolded form in 8 M urea. In each of these mutants, one of the residues of strand β9, from R572 to V579, was replaced by a cysteine and labeled with the fluorophore IAEDANS for site-directed fluorescence spectroscopy. Upon urea-dilution, the mutants folded into the native structure and were inserted into lipid bilayers of dilauroylphosphatidylcholine, similar to wild-type BamA. An aqueous and a membrane-adsorbed folding intermediate of BamA could be identified by strong shifts in the intensity maxima of the IAEDANS fluorescence of the labeled mutants of BamA towards shorter wavelengths, even in the absence of lipid bilayers. The shifts were greatest for membrane-adsorbed mutants and smaller for the inserted, folded mutants or the aqueous intermediates. The spectra of the mutants V573C-, L575C-, G577C-, and V579C-BamA, facing the lipid bilayer, displayed stronger shifts than the spectra recorded for the mutants R572C-, N574C-, T576C-, and K578C-BamA, facing the β-barrel lumen, in both the membrane-adsorbed form and the folded, inserted form. This alternating pattern was neither observed for the IAEDANS spectra of the unfolded forms nor for the water-collapsed forms, indicating that strand β9 forms in a membrane-adsorbed folding intermediate of BamA. The combination of cysteine scanning mutagenesis and site-directed fluorescence labeling is shown to be a valuable tool in examining the local secondary structure formation of transmembrane proteins. open access Herwig, Sascha Kleinschmidt, Jörg Helmut doi:10.3390/membranes13020247 Membranproteine Transmembranproteine Proteinfaltung Lipide Doppelschicht Protein OmpA Fluoreszenzspektroskopie publishedVersion eissn:2077-0375 Issue 2 Membranes Volume 13 false 247 </resource>
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