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Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy

A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to char...

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Autores principales: Werbeck, Nicolas D, Kirkpatrick, John, Reinstein, Jochen, Hansen, D Flemming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: WILEY-VCH Verlag 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016748/
https://www.ncbi.nlm.nih.gov/pubmed/24520048
http://dx.doi.org/10.1002/cbic.201300700
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author Werbeck, Nicolas D
Kirkpatrick, John
Reinstein, Jochen
Hansen, D Flemming
author_facet Werbeck, Nicolas D
Kirkpatrick, John
Reinstein, Jochen
Hansen, D Flemming
author_sort Werbeck, Nicolas D
collection PubMed
description A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the (1)H,(15)N spin-pair of enzyme-bound (15)NH(4)(+) can be probed by (15)N-edited heteronuclear NMR experiments. Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site. Ammonium activates both enzymes in a similar way to potassium, thus supporting this non-invasive approach. Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8. Our method for mapping the binding site is general and does not require chemical shift assignment of the enzyme resonances.
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spelling pubmed-40167482014-05-12 Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy Werbeck, Nicolas D Kirkpatrick, John Reinstein, Jochen Hansen, D Flemming Chembiochem Full Papers A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the (1)H,(15)N spin-pair of enzyme-bound (15)NH(4)(+) can be probed by (15)N-edited heteronuclear NMR experiments. Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site. Ammonium activates both enzymes in a similar way to potassium, thus supporting this non-invasive approach. Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8. Our method for mapping the binding site is general and does not require chemical shift assignment of the enzyme resonances. WILEY-VCH Verlag 2014-03-03 2014-02-12 /pmc/articles/PMC4016748/ /pubmed/24520048 http://dx.doi.org/10.1002/cbic.201300700 Text en © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. https://creativecommons.org/licenses/by/4.0/ © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Full Papers
Werbeck, Nicolas D
Kirkpatrick, John
Reinstein, Jochen
Hansen, D Flemming
Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title_full Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title_fullStr Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title_full_unstemmed Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title_short Using (15)N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
title_sort using (15)n-ammonium to characterise and map potassium binding sites in proteins by nmr spectroscopy
topic Full Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016748/
https://www.ncbi.nlm.nih.gov/pubmed/24520048
http://dx.doi.org/10.1002/cbic.201300700
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