Binding of metal ions toE. coli RNase HI observed by1H−15N heteronuclear 2D NMR
- Cite this article as:
- Oda, Y., Nakamura, H., Kanaya, S. et al. J Biomol NMR (1991) 1: 247. doi:10.1007/BF01875518
The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using1H−15N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide15N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg2+, Ca2+, or Ba2+. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (KD=2–4 mM for the current divalent ions).