title: 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.
authors.affiliation: Department of Biophysics, Free University, Amsterdam, The Netherlands.
authors.forename: M A
authors.initials: MA
authors.lastname: Scheerhagen
authors.forename: C A
authors.initials: CA
authors.lastname: Vlaanderen
authors.forename: J
authors.initials: J
authors.lastname: Blok
authors.forename: R
authors.initials: R
authors.lastname: van Grondelle
mesh_terms: D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D013604:T-Phages / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D014764:Viral Proteins