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TvoK is a prokaryotic K þ channel whose gating is modulated by divalent cation-binding to a carboxy-terminal RCK domain. To gain insight toward mechanisms underlying divalent cation binding and subsequent conformational changes, we measured chemical shift perturbations upon ligand binding in the soluble cytoplasmic RCK domain of TvoK using heteronuclear NMR spectroscopy.Uniformly 15 N-labeled, highly deuterated TvoK RCK domain was overexpressed in E.coli and purified by affinity and gel-filtration chromatography. 15 N-HSQC spectra showed well-dispersed crosspeaks corresponding to >85% of the 238 predicted backbone NH groups. Five-point titration experiments using 0 to 100 ˛1/4M Mn 2þ identified 12 residues that surrounded a putative divalent cation binding site, as indicated by spectral line-broadening due to the paramagnetic relaxation enhancement effect of Mn 2þ (Mn-PRE). Partial resonance assignments, made through a combination of HNCA experiments and residue-specific 15 N-labeling, identify D192 and E226 as key residues in divalent cation coordination, as indicated by high sensitivity to Mn-PRE (K app < 10 ˛1/4M). Further resonance assignments will identify remaining residues that lie within ~15 A ˚of the binding site. These experiments may reveal differences between the structural and chemical properties of the TvoK binding site and the Ca 2þ -selective binding site of the MthK RCK domain, which may underlie differential selectivities of MthK and TvoK RCK domains for divalent cations.