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Using 15 N‐Ammonium to Characterise and Map Potassium Binding Sites in Proteins by NMR Spectroscopy
Author(s) -
Werbeck Nicolas D.,
Kirkpatrick John,
Reinstein Jochen,
Hansen D. Flemming
Publication year - 2014
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300700
Subject(s) - chemistry , potassium , heteronuclear molecule , binding site , nuclear magnetic resonance spectroscopy , enzyme , ammonium , active site , stereochemistry , biochemistry , organic chemistry
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.