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 protein tryptophan fluorescence. It results from the efficient fluorescence energy transfer (E) from tryptophans to analogs bound in the nucleotide-binding site, indicating that protein tryptophans are "clustered" in close proximity to the binding site. This is in contrast to the lack of detectable energy transfer to the fluorescent single-stranded 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 bound nucleotides/DnaB hexamer is nonlinear, implying a larger separation between tryptophans and the bound nucleotide in the low affinity sites. Spectroscopic studies reveal that tryptophan residues are located on the surface of the DnaB helicase in a hydrophobic cleft, whereas the environment of the tyrosines is heterogeneous, with 6 out of 10 tyrosine 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, possibly reflecting the separation of the nucleotide- and ssDNA-binding sites, with tyrosines constituting part of the ssDNA-binding region.
Bujalowski
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Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555-0653.
Klonowska
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D020106:Acrylamide; D000178:Acrylamides / Q000737:chemistry; D000227:Adenine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000648:ultrastructure*; D001665:Binding Sites; D004265:DNA Helicases / Q000648:ultrastructure*; D004261:DNA Replication; D053488:DnaB Helicases; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D066298:In Vitro Techniques; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry
D000178:Acrylamides; D000227:Adenine Nucleotides; D001426:Bacterial Proteins; D020106:Acrylamide; D014364:Tryptophan; D004265:DNA Helicases; D053488:DnaB Helicases