Dynamic Bending Rigidity of a 200-bp DNA in 4 mM Ionic Strength: A Transient Polarization Grating Study

• Published in: Biophysical journal 2000-03, Vol.78 (3), p.1498-1518
• Main Authors: Naimushin, Alexei N., Fujimoto, Bryant S., Schurr, J. Michael
• Format: Article
• Language: English
• Subjects: Base Pairing; DNA - chemistry; Elasticity; Ethidium; Fluorescence Polarization; Kinetics; Methylene Blue; Models, Theoretical; Nucleic Acid Conformation; Osmolar Concentration
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(00)76703-8

DNA may exhibit three different kinds of bends: 1) permanent bends; 2) slowly relaxing bends due to fluctuations in a prevailing equilibrium between differently curved secondary conformations; and 3) rapidly relaxing dynamic bends within a single potential-of-mean-force basin. The dynamic bending rigidity ( κ d), or equivalently the dynamic persistence length, P d = κ d /k B T, governs the rapidly relaxing bends, which are responsible for the flexural dynamics of DNA on a short time scale, t ≤ 10 −5 s. However, all three kinds of bends contribute to the total equilibrium persistence length, P tot, according to 1/P tot ≅ 1/P pb + 1/P sr + 1/P d, where P pb is the contribution of the permanent bends and P sr is the contribution of the slowly relaxing bends. Both P d and P tot are determined for the same 200-bp DNA in 4 mM ionic strength by measuring its optical anisotropy, r( t) , from 0 to 10 μs. Time-resolved fluorescence polarization anisotropy (FPA) measurements yield r( t) for DNA/ethidium complexes (1 dye/200 bp) from 0 to 120 ns. A new transient polarization grating (TPG) experiment provides r( t) for DNA/methylene blue complexes (1 dye/100 bp) over a much longer time span, from 20 ns to 10 μs. Accurate data in the very tail of the decay enable a model-independent determination of the relaxation time ( τ R) of the end-over-end tumbling motion, from which P tot = 500 Å is estimated. The FPA data are used to obtain the best-fit pairs of P d and torsion elastic constant ( α) values that fit those data equally well, and which are used to eliminate α as an independent variable. When the relevant theory is fitted to the entire TPG signal ( S( t)) , the end-over-end rotational diffusion coefficient is fixed at its measured value and α is eliminated in favor of P d. Neither a true minimum in chi-squared nor a satisfactory fit could be obtained for P d anywhere in the range 500–5000 Å, unless an adjustable amplitude of azimuthal wobble of the methylene blue was admitted. In that case, a well-defined global minimum and a reasonably good fit emerged at P d = 2000 Å and 〈 δζ 2〉 1/2 = 25°. The discrimination against P d values