Abstract
A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ldT, dT)n and (lA,A)n, which served as macro-molecular spin probes in ESR competition experiments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity domain. In 20 mM Tris X HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton, and 125 mM NaCl, the following affinity relationship was observed: K(dT)napp = 10(3) KfdDNAapp = 2 X 10(4) K(A)napp = 6.6 X 10(4) KrRNAapp = 1.5 X 10(5) KR17RNAapp. Increasing the [NaCl] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (lA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kint, of 1300 M-1 and a cooperativity factor, omega, of 60 (Kint omega = Kapp) for (lA,A)n.
Highlights
Titative determination of nucleic acid binding affinities The high binding affinities often present inprotein-nucleic of proteins under physiologically relevant conditions acid interactions have caused serious problems when analyzhas been applied to the gene5 protein of bacteriophage ing such interactions by conventional physical methods
This was achieved with two spin-labeled nucleic order to better understand the fundamenrteaclognition procacids, (ZdT,dT), and (ZA,A)n,which served as macromolecular spin probes in ESR competition experiments
A put considerable effort in designing a sensitivEe SR approach computer fit of theexperimental titration data at 200 which allows a direct quantitative determination of relative mM NaCl gave an intrinsic binding constant, Kin, of nucleic acid binding affinitiesof proteins binding under phys
Summary
We reported earlier the ESR line shapes of (IdT,dT), in the presence and absenceof gene 5protein [9] as well as plots. For binding of the gene 5 protein to (lA,A),,.By utilizing the entire digitized ESR spectral arrays measured during the titration of (lA,A), with gene 5 protein,it was foundthat atwocomponentanalysismethodcan beused to determine the fraction F of complexed spin-labeled polynucleotides as was that purpose, the initial slope which is the samefor both salt concentrations is extrapolated to F = 1, and the projected value of the abscissa is taken for the [protein]/[A] ratio. It follows t h a t gene 5 protein covers about 4 nucleotide residues, a valuewhich is in good agreement with titrationresults obtained earlierwith (IdT,dT), aswell as with other spectrothe case for bacteriophage T4 gene 32 protein and (IdT,dT), scopic techniques.
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