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The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli

The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of...

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Autores principales: Hermansen, Simen, Ryoo, David, Orwick-Rydmark, Marcella, Saragliadis, Athanasios, Gumbart, James C., Linke, Dirk
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9329534/
https://www.ncbi.nlm.nih.gov/pubmed/35911955
http://dx.doi.org/10.3389/fmolb.2022.918480
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author Hermansen, Simen
Ryoo, David
Orwick-Rydmark, Marcella
Saragliadis, Athanasios
Gumbart, James C.
Linke, Dirk
author_facet Hermansen, Simen
Ryoo, David
Orwick-Rydmark, Marcella
Saragliadis, Athanasios
Gumbart, James C.
Linke, Dirk
author_sort Hermansen, Simen
collection PubMed
description The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo, but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.
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spelling pubmed-93295342022-07-29 The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli Hermansen, Simen Ryoo, David Orwick-Rydmark, Marcella Saragliadis, Athanasios Gumbart, James C. Linke, Dirk Front Mol Biosci Molecular Biosciences The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo, but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display. Frontiers Media S.A. 2022-07-14 /pmc/articles/PMC9329534/ /pubmed/35911955 http://dx.doi.org/10.3389/fmolb.2022.918480 Text en Copyright © 2022 Hermansen, Ryoo, Orwick-Rydmark, Saragliadis, Gumbart and Linke. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Hermansen, Simen
Ryoo, David
Orwick-Rydmark, Marcella
Saragliadis, Athanasios
Gumbart, James C.
Linke, Dirk
The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title_full The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title_fullStr The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title_full_unstemmed The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title_short The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli
title_sort role of extracellular loops in the folding of outer membrane protein x (ompx) of escherichia coli
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9329534/
https://www.ncbi.nlm.nih.gov/pubmed/35911955
http://dx.doi.org/10.3389/fmolb.2022.918480
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