Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure.

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.

A model for the complex between the helix destabilizing protein GP32 of bacteriophage T4 and single-stranded DNA.

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.

Binding stoichiometry of the gene 32 protein of phage T4 in the complex with single stranded DNA deduced from boundary sedimentation.

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.

Hydrodynamic studies of a DNA-protein complex. Dimensions of the complex of single-stranded 145 base DNA with gene 32 protein of phage T4 deduced from quasi-elastic light scattering.

The translational diffusion coefficient of the saturated complex of single-stranded 145 base DNA and the helix-destabilizing protein of phage T4, GP32, can be measured at equilibrium by means of quasi-elastic light scattering. If the complex is considered as a rigid rod one can estimate its dimensions by combining the translational diffusion coefficient with earlier data on rotational diffusion. It was found that the average base-base distance of the 145 base DNA in the complex is between 4.3 and 4.7 A, while the diameter of the complex is between 44 and 68 A. This suggests that the conformation of the complex must be such that a large amount of water is trapped.

The conformation of the complex of the helix destabilizing protein GP32 of bacteriophage T4 and single stranded DNA.

The conformation of single stranded polynucleotides is changed specifically upon binding of the helix destabilizing protein of bacteriophage T4 (GP32). On the basis of circular dichroism (CD) and absorption experiments it is shown that denaturing conditions and the binding of oligopeptides can not induce the altered conformation. On the contrary, according to the current CD and absorption theory, the optical properties of the complex can be explained by a specific, regular conformation, characterized by an appreciable tilt of the bases (less than or equal to -10 degrees) and either a small rotation per base or a small helix diameter. This conformation agrees nicely with the increase of the base-base distance in the complex as determined in solution by electric field induced birefringence measurements. Our calculations show that also the model proposed by Alma (Ph.D. Thesis Catholic University Nijmegen, The Netherlands (1982)) for the complex of the helix destabilizing protein of bacteriophage fd, in which the helix diameter is large and the bases are almost parallel to the helix axis, would agree with the CD- and absorption spectra of the GP32-complex. For the latter protein this model would have to be modified with regard to the axial increment of the bases which is much larger in the GP32-complexes.

Hydrodynamic studies of a DNA-protein complex. Elongation of single stranded nucleic acids upon complexation with the gene 32 protein of phage T4 deduced from electric field-induced birefringence experiments.

Short DNA and RNA fragments complexed with the helix destabilizing protein of bacteriophage T4, GP32, have been studied in solution by electric birefringence and circular dichroism. The birefringence of the complexes is positive and the magnitude indicates that the DNA and RNA fragments become linear and rigid upon protein binding. The field free decay is biphasic. On the basis of a rigid rod approximation the slow relaxation time leads to a base-base distance along the helix axis in the complex from 4.3 to 5.6 A, an elongation of at least 50% compared to single-stranded DNA.