Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system.
ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro.
Imperial
J
J
Department of Nutritional Sciences, University of California-Berkeley, Berkeley, CA 94720, USA. jimperial@bit.etsia.upm.es
Hadi
M
M
Amy
N K
NK
D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008982:Molybdenum / Q000378:metabolism*; D008983:Molybdoferredoxin / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D011869:Radioligand Assay; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017973:Tungsten Compounds / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C098002:ModA protein, E coli; D008983:Molybdoferredoxin; D033902:Periplasmic Binding Proteins; D017973:Tungsten Compounds; C044659:molybdate; D008982:Molybdenum; C045951:tungstate