Hydrodynamic studies of a DNA‐protein complex

Scheerhagen, M. A.; Amerongen, Herbert van; Grondelle, Rienk van; Blök, J.

doi:10.1016/0014-5793(85)80522-6

Cited by 16 publications

(17 citation statements)

References 10 publications

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“…Both numbers are in good agreement with the value obtained from the experiments with the 145 b DNA complex. Since it may be expected that the size of the binding site of GP32 is independent of the polynucleotide, these results support the relatively large axial increment in the tRNA-GP32 complex obtained from birefringence experiments ( 13).…”

Section: M=-supporting

confidence: 51%

“…In our opinion this is mainly due to the unpredictable variability in the properties of the biological components. Comparable variations were observed in the ELB-and QELS-experiments ( 13,14). For the 145 b-complex the sedimentation coefficient at 50 mM NaCl was measured at various temperatures (5°-22°C) and binding ratios (2.5-12.5 nucl./protein).…”

Section: Measurement Of Sedimentation Coefficientsmentioning

confidence: 78%

“…The experimental set-up for the electric field induced birefringence experiments has been described elsewhere ( 13).…”

Section: Methodsmentioning

confidence: 99%

“…However, the rotational diffusion coefficient has been determined from the decay of electric birefringence (13) and the diameter of the 145 b DNA complex was estimated to be 60 A (14). If it is assumed that the diameter of the tRNA complex, d, has roughly the same magnitude, e.g.…”

Section: M=-mentioning

confidence: 99%

“…Recently, we have reported two hydrodynamic studies on the complex of GP32 and ss DNA with 145 bases ( 13,14). It was shown that the properties of saturated complexes could be very well studied in solution under equilibrium conditions.…”

Section: Introductionmentioning

confidence: 99%

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

Scheerhagen¹,

Vlaanderen²,

Blök³

et al. 1986

Journal of Biomolecular Structure and Dynamics

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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.

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Section: M=-supporting

confidence: 51%

Section: Measurement Of Sedimentation Coefficientsmentioning

confidence: 78%

“…The experimental set-up for the electric field induced birefringence experiments has been described elsewhere ( 13).…”

Section: Methodsmentioning

confidence: 99%

Section: M=-mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Scheerhagen¹,

Vlaanderen²,

Blök³

et al. 1986

Journal of Biomolecular Structure and Dynamics

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A specific model for the conformation of single‐stranded polynucleotides in complex with the helix‐destabilizing protein GP32 of bacteriophage T4

Scheerhagen¹,

Bokma²,

Vlaanderen³

et al. 1986

Biopolymers

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SynopsisThe CD and absorption (OD) spectra of single-stranded nucleic acids in complex with the helix-destabilizing protein of either bacteriophage T4 (GP32) or bacteriophage fd (GP5) show similar and unusual features for all polynucleotides investigated. The change in the CD spectra between 310 and 240 nm is in all cases characterized by a considerable decrease in the positive band, while the negative band (if present) remains relatively intense. These changes are different from those due to temperature or solvent denaturation and, moreover, cannot be induced by the binding of simple oligopeptides. Absorption measurements show that all polynucleotides remain hypochromic in the complex. Both CD and OD spectra point to a specific and probably similar conformation in complex for all polynucleotides with substantial interactions between the bases. The spectral properties are almost temperature independent (0-40°C). Therefore, we conclude that the conformation must be regular and rigid. To investigate the relation between these optical properties and the specific polynucleotide structure, CD and OD spectra were calculated for an adenine hexamer over a wide range of the conformational parameters. It appears that the calculated CD intensity is not very sensitive to an increase in the axial increment and that many different conformations can give rise to more or less similar CD spectra. However, simulation of the very nonconservative experimental CD spectrum of the poly(rA)-GP32 complex requires that the conformation satisfies two criteria: (1) a considerable tilt of the bases (< -10") in combination with (2) a small rotation per base (=ZOO) andor a position of the bases close to the helix axis (dx = 0 A). Such conformations can also explain the observed hyperchromism upon binding of GP32 to poly(rA)/(dA). Very similar structural characteristics also account for the optical properties of the complexes with GP5. These are discussed as an alternative to the structure suggested by Alma-Zeestraten for poly(dA) in the complex [N. C. M. Alma-Zeestraten (1982) Doctoral thesis Catholic University, Nijmegen, The Netherlands]. The secondary structure proposed in this work can be reconciled with the overall dimensions of the complex, assuming that the polynucleotide helix is further organized in a superhelix.

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