Metal-protein interactions are not necessarily tight in many transient biological processes, such as cellular signaling, enzyme regulation, and molecular recognition. Here, we analyzed the binding thermodynamics and characterized the structural effect of divalent metal ions, i.e. Mn2+, Zn2+, and Mg2+, to the isolated ribonuclease H (RNH) of human immunodeficiency virus (HIV) using isothermal titration calorimetry (ITC) and circular dichroism. The binding thermodynamics of Mg2+ to RNH was determined using competition ITC experiments, and the binding affinity of Mg2+ was found to be about 40- and 400-times lower than those of Mn2+ and of Zn2+, respectively. The structural analysis showed that Mg2+ binding had little effect on the thermal stability of RNH, while Zn2+ and Mn2+ binding increased the stability. The thermodynamic characteristics of RNH metal binding, compared to intact HIV reverse transcriptase, and a possible mechanism of conformational change induced upon metal ion binding, in correlation with the structure-function relationship, are discussed.
Keywords: RNase H; circular dichroism; co-factor; isothermal titration calorimetry; metal-protein interaction.