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Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

BACKGROUND: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthe...

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Autores principales: Krewinkel, Manuel, Dworeck, Tamara, Fioroni, Marco
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170585/
https://www.ncbi.nlm.nih.gov/pubmed/21854627
http://dx.doi.org/10.1186/1477-3155-9-33
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author Krewinkel, Manuel
Dworeck, Tamara
Fioroni, Marco
author_facet Krewinkel, Manuel
Dworeck, Tamara
Fioroni, Marco
author_sort Krewinkel, Manuel
collection PubMed
description BACKGROUND: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. RESULTS: The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. CONCLUSION: In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.
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spelling pubmed-31705852011-09-11 Engineering of an E. coli outer membrane protein FhuA with increased channel diameter Krewinkel, Manuel Dworeck, Tamara Fioroni, Marco J Nanobiotechnology Research BACKGROUND: Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. RESULTS: The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD) spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine) were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. CONCLUSION: In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications. BioMed Central 2011-08-19 /pmc/articles/PMC3170585/ /pubmed/21854627 http://dx.doi.org/10.1186/1477-3155-9-33 Text en Copyright ©2011 Krewinkel et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Krewinkel, Manuel
Dworeck, Tamara
Fioroni, Marco
Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title_full Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title_fullStr Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title_full_unstemmed Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title_short Engineering of an E. coli outer membrane protein FhuA with increased channel diameter
title_sort engineering of an e. coli outer membrane protein fhua with increased channel diameter
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3170585/
https://www.ncbi.nlm.nih.gov/pubmed/21854627
http://dx.doi.org/10.1186/1477-3155-9-33
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