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 beta-roll structure and bind Ca(2+) (one ion per repeat). To gain insight into NodO's secondary and tertiary structures, and their dependence upon Ca(2+) binding, we performed fluorescence experiments and FTIR spectroscopy. We found that calcium binds to the protein, promoting about a 10% increase in beta-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. Tb(3+), a trivalent lanthanide, can compete with Ca(2+) for the same binding sites, but with higher affinity. The number of Ca(2+) binding sites, estimated by FTIR spectroscopy, was found to be consistent with the number of predicted repeats.
Serra
M D
MD
Centro ITC-CNR Fisica Stati Aggregati, Via Sommarive 18, Povo (Trento), I-38050, Italy.
Sutton
J M
JM
Höper
F
F
Downie
J A
JA
Menestrina
G
G
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017433:Protein Structure, Secondary; D011522:Protons; D016961:Rhizobium leguminosarum / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013725:Terbium / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; C062095:NodO protein, Rhizobium leguminosarum; D011522:Protons; D013725:Terbium; C027132:1-anilino-8-naphthalenesulfonate; D002118:Calcium