Effects of Calcium and Protons on the Secondary Structure of the Nodulation Protein NodO from Rhizobium leguminosarum biovar viciae

NodO, a 30-kDa nodulation protein secreted by Rhizobium leguminosarum biovar viciae, belongs to a family of proteins produced by Gram-negative bacteria containing a variable number of glycine/aspartates nonapeptides. In some instances, these are organized into a parallel β-roll structure and bind Ca2+ (one ion per repeat). To gain insight into NodO's secondary and tertiary structures, and their dependence upon Ca2+ binding, we performed fluorescence experiments and FTIR spectroscopy. We found that calcium binds to the protein, promoting about a 10% increase in β-structure mainly to the expense of random-coil. Protons can also induce a reversible change in NodO structure, as indicated by quenching of intrinsic tryptophan fluorescence and binding of ANS, albeit probably via a different mechanism. Tb3+, a trivalent lanthanide, can compete with Ca2+ for the same binding sites, but with higher affinity. The number of Ca2+ binding sites, estimated by FTIR spectroscopy, was found to be consistent with the number of predicted repeats.