Spectroscopic demonstration of a linkage between the kinetics of NTP hydrolysis and the conformational state of the recA-single-stranded DNA complex
| Autor: | Floyd R. Bryant, Einar Stole |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
Stereochemistry
Mutant DNA Single-Stranded Biochemistry Cofactor chemistry.chemical_compound Structure-Activity Relationship Point Mutation Histidine Molecular Biology Purine Nucleotides Quenching (fluorescence) Binding Sites biology Tryptophan Cell Biology Hydrogen-Ion Concentration Fluorescence Recombinant Proteins Kinetics Rec A Recombinases Spectrometry Fluorescence chemistry biology.protein Nucleoside triphosphate Nucleoside DNA |
| Zdroj: | The Journal of biological chemistry. 270(35) |
| ISSN: | 0021-9258 |
| Popis: | We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation. |
| Databáze: | OpenAIRE |
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