A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed...

Descripción completa

Guardado en:
Detalles Bibliográficos
Autores principales: Jennifer Mehlhorn, Helena Steinocher, Sebastian Beck, John T M Kennis, Peter Hegemann, Tilo Mathes
Formato: article
Lenguaje:EN
Publicado: Public Library of Science (PLoS) 2013
Materias:
R
Q
Acceso en línea:https://doaj.org/article/aee3a6e42e1344e9b8a985f9dc7b1a39
Etiquetas: Agregar Etiqueta
Sin Etiquetas, Sea el primero en etiquetar este registro!
id oai:doaj.org-article:aee3a6e42e1344e9b8a985f9dc7b1a39
record_format dspace
spelling oai:doaj.org-article:aee3a6e42e1344e9b8a985f9dc7b1a392021-11-18T08:48:43ZA set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.1932-620310.1371/journal.pone.0079006https://doaj.org/article/aee3a6e42e1344e9b8a985f9dc7b1a392013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24223875/?tool=EBIhttps://doaj.org/toc/1932-6203Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.Jennifer MehlhornHelena SteinocherSebastian BeckJohn T M KennisPeter HegemannTilo MathesPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e79006 (2013)
institution DOAJ
collection DOAJ
language EN
topic Medicine
R
Science
Q
spellingShingle Medicine
R
Science
Q
Jennifer Mehlhorn
Helena Steinocher
Sebastian Beck
John T M Kennis
Peter Hegemann
Tilo Mathes
A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
description Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.
format article
author Jennifer Mehlhorn
Helena Steinocher
Sebastian Beck
John T M Kennis
Peter Hegemann
Tilo Mathes
author_facet Jennifer Mehlhorn
Helena Steinocher
Sebastian Beck
John T M Kennis
Peter Hegemann
Tilo Mathes
author_sort Jennifer Mehlhorn
title A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
title_short A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
title_full A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
title_fullStr A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
title_full_unstemmed A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
title_sort set of engineered escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.
publisher Public Library of Science (PLoS)
publishDate 2013
url https://doaj.org/article/aee3a6e42e1344e9b8a985f9dc7b1a39
work_keys_str_mv AT jennifermehlhorn asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT helenasteinocher asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT sebastianbeck asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT johntmkennis asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT peterhegemann asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT tilomathes asetofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT jennifermehlhorn setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT helenasteinocher setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT sebastianbeck setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT johntmkennis setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT peterhegemann setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
AT tilomathes setofengineeredescherichiacoliexpressionstrainsforselectiveisotopeandreactivitylabelingofaminoacidsidechainsandflavincofactors
_version_ 1718421282026946560