CLOSE PROXIMITY OF TRYPTOPHAN RESIDUES AND ATP-BINDING SITE IN ESCHERICHIA-COLI PRIMARY REPLICATIVE HELICASE DNAB PROTEIN - MOLECULAR TOPOGRAPHY OF THE ENZYME

The binding of fluorescent nucleotide analogs to the Escherichia coli primary replicative helicase DnaB protein causes strong quenching of p rotein tryptophan fluorescence. It results from the efficient fluoresc ence energy transfer (E) from tryptophans to analogs bound in the nucl eotide-binding site, indicating that protein tryptophans are ''cluster ed'' in close proximity to the binding site. This is in contrast to th e lack of detectable energy transfer to the fluorescent single strande d DNA (ssDNA) derivative, suggesting a distant separation between two function-linked structural elements of the enzyme, the nucleotide- and ssDNA-binding sites. The dependence of E upon the average number of b ound nucleotides/DnaB hexamer is nonlinear, implying a larger separati on between tryptophans and the bound nucleotide in the low affinity si tes. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas t he environment of the tyrosines is heterogeneous, with 6 out of 10 tyr osine residues located on the surface of the helicase. The efficiency of the fluorescence energy transfer from the tyrosines to tryptophans suggests that the ''centers of mass'' of the residues are separated, p ossibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA binding region.