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: Next, we tested whether it was possible to observe binding of 15NH4+ to HDAC8 by NMR. As expected, bulk 15NH4+ was not observed in 15N-edited, proton-detected 1D NMR spectra in buffer containing 200 mm15NH4+ at pH 8.0 (Figure 2 A) because of rapid exchange of the ammonium protons with the bulk solvent. The 15N chemical shift of the free ammonium was therefore determined by direct 15N-detection to be 20.5 ppm (Figure 2 A, inset). Addition of [14N]HDAC8, however, resulted in a distinct peak in the 15N-edited experiment, indicative of HDAC8-bound 15NH4+ with a proton chemical shift of 7.1 ppm (Figure 2 B). A 15N chemical shift of 25.5 ppm was subsequently determined by recording a 2D 1H,15N HSQC spectrum (Figure 2 C).
Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). email@example.com.