Fluorescence spectroscopy structural studies of Bp-UreG in solution

The nickel-enzyme urease is a α3β3γ3 complex that catalyses the hydrolysis of urea in the last step of nitrogen mineralization. Full catalytic function requires the interaction of four accessory proteins: UreD, UreF, UreG and UreE. The monomer form of UreG (23kD) contains a fully conserved P-loop nucleotide-binding motif and has been proposed to be essential, as UreDFG complex, in coupling cellular metabolism and the assembly of urease. Here, the different intrinsic fluorescence properties of the single tryptophan residue (W192) of the UreG accessory protein from B. pasteurii have been analyzed to provide structural information. Under native conditions, at 20°C in 20 mM phosphate buffer, pH 7.5, BpUreG exists predominantly as a dimer with the emission maximum at 336 nm, an anisotropy decay correlation time, φ, of 32 ns and a KI collisional quenching rate constant, kq, of 0.35x109sec-1M-1. In the presence of 1.8 M Gdn-HCl, the emission maximum is shifted to 347 nm with an increase of 10% of the fluorescence intensity. Under the same condition an anisotropy decay correlation time, φ, of 11 ns and a KI collisional quenching rate constant, kq, of 0.99x109sec-1M-1 were recovered. In addition, fluorescence-detected denaturation curves revealed that the tryptophan peptide chain is fully unfolded at 2 M Gdn-HCl. Altogether our results indicate the existence of a relatively unstable region at the C-terminus of BpUreG with the tryptophan-containing segment that can move from a buried to an exposed configuration. Loss of local structure can be concomitant with dimer dissociation, but is apparently unrelated to the folding state of the residual protein core. This work was supported with a MIUR-PRIN 2003 to S.L.C.