Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.
Bottom Line: 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.
Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). firstname.lastname@example.org.Show MeSH
Mentions: Another example of a potassium-binding enzyme is the molecular chaperone Hsp70. Two potassium ions in the ATP-binding domain (Figure 3 A) have been identified to be crucial for the ATPase cycle,[6b] which in turn regulates the binding and release of substrate proteins. In a similar way to that for HDAC8, these potassium ions can be replaced by ammonium, thereby resulting in approximately half of the ATPase activity observed with potassium.[6b] Based on our success in characterising the potassium binding sites of HDAC8 with 15NH4+, we tested the general applicability of our approach by probing the potassium binding sites of the 41 kDa ATP-binding domain of DnaK, a bacterial Hsp70 homologue from Thermus thermophilus.
Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). email@example.com.