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.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 24 Sep 1999|
Bibliographical noteFunding Information:
This work was financially supported by the Italian Consiglio Na-zionale delle Ricerche (CNR), by the Istituto Trentino di Cultura (ITC), by a special grant from the Provincia Autonoma di Trento (PAT, 1913/CONV/1458), and by the British Biotechnology and Biological Sciences Research Council (BBSRC). MDS was the recipient of a fellowship from CNR (N° 201.02.45-21.02.05), FH was supported by a post-doctoral fellowship of the European Community (European Project CRHX-CT93-055), and JMS was employed on BBSRC Grant PG208/554.