Werbeck, ND;
Kirkpatrick, J;
Reinstein, J;
Hansen, DF;
(2014)
Using (15) N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy.
Chembiochem
, 15
(4)
pp. 543-548.
10.1002/cbic.201300700.
![[thumbnail of 543_ftp.pdf]](https://discovery.ucl.ac.uk/style/images/fileicons/application_pdf.png) Preview |
PDF
543_ftp.pdf Download (1MB) |
Abstract
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) NH4 (+) 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.
| Type: | Article |
|---|---|
| Title: | Using (15) N-Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy. |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1002/cbic.201300700 |
| Publisher version: | http://dx.doi.org/10.1002/cbic.201300700 |
| Additional information: | � 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. |
| Keywords: | HDAC, Hsp70, NMR spectroscopy, ammonium, enzyme catalysis, potassium binding |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1426601 |
Loading...
Loading...Loading...Loading...Archive Staff Only
 |
View Item |
