Binding Stoichiometry of the Gene 32 Protein of Phage T4 in the Complex with Single Stranded DNA Deduced from Boundary Sedimentation

Abstract

Short 145 base DNA fragments in complex with the helix destabilizing protein of bacteriophage T4, GP32, have been studied with boundary sedimentation. The sedimentation coefficient was determined as a function of concentration, protein-nucleic acid ratio, temperature and salt concentration. It can be concluded that the measured values reflect the properties of the saturated DNA-GP32 complex. A combination of the earlier obtained translational diffusion coefficient of the complex with the sedimentation coefficient yields its anhydrous molecular weight (Mw = 5.4.10(5) D), which corresponds to a size of the binding site of 10 nucleotides per protein. This procedure is not sensitive to the presence of non-binding protein molecules and to the assumed protein concentration, and therefore, it seems more reliable than a determination from titration experiments. Similar sedimentation measurements were performed with tRNA-complexes containing 76 nucleotides. The translational diffusion coefficient can be calculated from the measured rotational diffusion coefficient and assuming the same hydrodynamic diameter for this complex as obtained for the 145 b DNA complex. The molecular weight derived from the data then also leads to a binding site size of about 10 nucleotides. This suggests that also the short tRNA-complex forms an open, strongly solvated structure, as was proposed for the 145 b DNA-GP32 complex.

Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase

“…shows strand-exchange reactions performed with either wild-type, H195A, or H195Q UvsX at a concentration of 3 μM (a 1.2-fold excess over ssDNA-binding sites), all in the presence of 1.43 μM Gp32 (exactly saturating with respect to ssDNA-binding sites, assuming that n =7 nucleotide residues/protomer27,28). Under these conditions, the reaction with wild-type UvsX was very robust, with nearly all replicative fragment III (RFIII; linearized M13mp18 dsDNA) substrates converted into high-molecular-weight branched DNA products by the 2-min time point (Fig.…”

Complex formation between the adenovirus DNA-binding protein and single-stranded poly(rA). Cooperativity and salt dependence

“…In conclusion, AdDBP binding to poly(rA) is characterized by a weak cooperativity ( > = 20-30) and a moderately strong, but salt-dependent binding constant. Quantitative analysis of the titration curves has shown that n-13 is a better estimate for the size of the binding site than = 9-11 (van Amerongen et al, 1987), although this number must still be considered a lower limit (Scheerhagen et al, 1986b). Binding of AdDBP to poly(dT) is probably very strong, yielding a close to stoichiometric binding and a "maximal" size of the binding site, = 15 (van Amerongen et al, 1987), rather similar to the improved value of = 13 for binding to poly(rA).…”

Interaction between adenovirus DNA-binding protein and single-stranded polynucleotides studied by circular dichroism and ultraviolet absorption

“…The determined value for n is a lower bound, because of the implicit assumption that all protein molecules can bind the polynucleotide and no protein is inactivated. CD titrations with GP32 and a number of polynucleotides systematically resulted in a value for n that is 10-20% too low (Scheerhagen et al, 1986b). The value of -9-11 is larger than the value of = 7 determined by sedimentation experiments (van der Vliet et al, 1978).…”