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 tran sport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescen ce emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA sho wed an intrinsic fluorescence emission spectrum attributable to its th ree tryptophanyl residues. Molybdate binding caused a conformational c hange in the protein characterized by: (i) a shift of tryptophanyl gro ups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at DH 7.8) of fluorescence; and (iii) a higher availabil ity of tryptophanyl groups to the polar quencher acrylamide, The tight binding of molybdate did not allow an accurate estimation of the bind ing constants by these indirect methods. An isotopic binding method wi th (MoO42-)-Mo-99 used for accurate determination of K-D (20 nM) and s toichiometry (1:1 molar ratio). ModA bound tungstate with approximatel y the same affinity, but did not bind sulfate or phosphate. These K(D) s are 150- to 250-fold lower than those previously reported, and compa tible with the high molybdate transport affinity of the mod system. Th e affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro. (C) 1998 Elsevier Science B.V.