Abstracts of 2000 Publications


Condensation of DNA by Multivalent Cations: Experimental Studies of Condensation Kinetics
He, S., P.G. Arscott, and V.A. Bloomfield (2000) , Biopolymers 53: 329-341. pdf

DNA in viruses and cells exists in highly condensed, tightly packaged states. We have undertaken an in vitro study of the kinetics of DNA condensation by the trivalent cation hexaammine cobalt (III) with the aim of formulating a quantitative, mechanistic model of the condensation process. Experimental approaches included total intensity and dynamic light scattering, electron microscopy, and differential sedimentation. We determined the average degree of condensation, the distribution of condensate sizes, and the fraction of uncondensed DNA as a function of reaction time for a range of [DNA] and [Co(NH3)(6)(3+)]. We find the following: (1) DNA condensation occurs only above a critical [Co(NH3)(6)(3+)] for a given DNA and salt concentration. At the onset of condensation, [Co(NH3)(6)(3+)]/[DNA-phosphate] is close to the average value of 0.54, which reflects the 89-90% charge neutralization criterion for condensation. (2) The equilibrium weight average hydrodynamic radius [R-H] of the condensates first decreases, then increases vith increasing [Co(NH3)(6)(3+)] as they undergo a transition from intramolecular (monomolecular): to intermolecular (multimolecular) condensation, However, [R-H] is insensitive to [DNA]. (3) The uncondensed DNA fraction decays approximately exponentially with rime. The equilibrium uncondensed DNA fraction and relaxation time decrease with increasing [Co(NH3)(6)(3+)] but are insensitive to [DNA]. (4) The condensation rate in its early stages is insensitive to [DNA] but proportional to [Co(NH3)(6)(3+)](xs) = [Co(NH3)(6)(3+)] - [Co(NH3)(6)(3+)](crit). (5) Data for low [DNA] and low [Co(NH3)(6)(3+)] at early stages of condensation are most reliable for kinetic modeling since under these conditions there is minimal clumping and network formation among separate condensates. A mechanism with initial monomolecular nucleation and subsequent bimolecular association and unimolecular dissociation steps with rate constants that depend on the number of DNA molecules in the condensate, accounts reasonably well for these observations.

Isothermal Titration Calorimetry and Electrostatic Mechanism
D. Matulis, I. Rouzina, and V.A. Bloomfield (2000) , J. Mol. Biol. 296: 1053-1063. pdf

The thermodynamics of binding of the trivalent cations cobalt hexammine and spermidine to plasmid DNA was studied by isothermal titration calorimetry. Two stages were observed in the course of titration, the first attributed to cation binding and the second to DNA condensation. A standard calorimetric data analysis was extended by applying an electrostatic binding model, which accounted for most of the observed data. Both the binding and condensation reactions were entropically driven (T Delta S similar to + 10 kcal/mol cation) and enthalpically opposed (Delta H similar to + 1 kcal/mol cation). As predicted from their relative sizes, the binding constants of the cations were indistinguishable, but cobalt hexammine had a much greater DNA condensing capacity because it is more compact than spermidine. The dependence of both the free energy of cobalt hexammine binding and the critical. cobalt hexammine concentration for DNA condensation on temperature and monovalent cation concentration followed the electrostatic model quite precisely. The heat capacity changes of both stages were positive, perhaps reflecting both the temperature dependence of the dielectric constant of water and the burial of polar surfaces. DNA condensation occurred when about 67% of the DNA phosphate charge was neutralized by cobalt hexammine and 87% by spermidine. During condensation, the remaining DNA charge was neutralized.

Stretching of Single Collapsed DNA Molecules
C.G. Baumann, V.A. Bloomfield, S.B. Smith, C. Bustamante, M.D. Wang, and S.M. Block (2000), Biophys. J., 78: 1965-1978. pdf

The elastic response of single plasmid and lambda phage DNA molecules was probed using optical tweezers at concentrations of trivalent cations that provoked DNA condensation in bulk. For uncondensed plasmids, the persistence length, P, decreased with increasing spermidine concentration before reaching a limiting value 40 nm. When condensed plasmids were stretched, two types of behavior were observed: a stick-release pattern and a plateau at similar to 20 pN. These behaviors are attributed to unpacking from a condensed structure, such as coiled DNA. Similarly, condensing concentrations of hexaammine cobalt(III) (CoHex) and spermidine induced extensive changes in the tow and high force elasticity of lambda DNA. The high force (5-15 pN) entropic elasticity showed worm-like chain (WLC) behavior, with P two- to fivefold lower than in low monovalent salt. At lower forces, a 14-pN plateau abruptly appeared. This corresponds to an intramolecular attraction of 0.083-0.33 kT/bp, consistent with osmotic stress measurements in bulk condensed DNA. The intramolecular attractive force with CoHex is larger than with spermidine, consistent with the greater efficiency with which CoHex condenses DNA in bulk. The transition from WLC behavior to condensation occurs at an extension about 85% of the contour length, permitting looping and nucleation of condensation. Approximately half as many base pairs are required to nucleate collapse in a stretched chain when CoHex is the condensing agent.

Static and Dynamic Light Scattering from Aggregating Particles
Bloomfield, V.A. (2000), Biopolymers 54: 168-172. pdf

We use standard hydrodynamic and light scattering theories to calculate the total intensity and dynamic light scattering properties of random aggregates of spherical particles containing up to ten spheres. When the aggregates have dimensions comparable to the wavelength of light, intraaggregate interference effects con dramatically reduce the apparent size of the aggregates. These results could be significant interpreting DNA condensation, protein polymerization, and other biomolecular aggregation reactions.

Tracer Diffusion of Proteins in DNA Solutions. 2. Green Fluorescent Protein in Crowded DNA Solutions
Busch, N.A., T. Kim, and V.A. Bloomfield (2000), Macromolecules 33: 5932-5937. pdf

Proteins diffuse to their sites of action within cells in a crowded, strongly interacting environment of nucleic acids and other macromolecules. To investigate the dynamics of a typical globular protein in such an environment, we used fluorescence photobleaching recovery to measure the probe diffusion of green fluorescent protein (GFP) in dilute to highly concentrated aqueous solutions of glycerol, Ficoll 70, and persistence-length calf thymus DNA. In glycerol, GFP accurately obeyed the Stokes-Einstein equation that relates diffusion coefficient to solution viscosity. In concentrated Ficoll 70, GFP diffused moderately faster than predicted from viscosity, demonstrating the phenomenon of microviscosity in a molecularly heterogeneous solution. In DNA, the diffusion coefficient of GFP was markedly greater than predicted from the Stokes-Einstein equation, with deviations increasing at lower ionic strength. This behavior reflects microviscosity, coulombic interactions and the dynamics of probe diffusion in DNA solutions that had undergone the ordinary--extraordinary transition, which we demonstrated by dynamic light scattering.

The Greater Negative Charge Density of DNA in Tris-Borate Buffers Does Not Enhance DNA Condensation by Multivalent Cations
J.J. Schwinefus and V.A. Bloomfield (2000), Biopolymers 54: 572-577. pdf

As indicated by recent measurements of the electrophoretic free solution mobility, DNA appears to have a 20% greater helical charge density in Tris-borate-EDTA (TBE) buffers than in Tris-acetate-EDTA (TAE) buffers. Since electrostatic forces play a major role in DNA packaging processes, we have investigated the condensation of closed circular plasmid DNA using total intensity and dynamic light scattering in Tris-borate, Tris-acetate, and Tris-cacodylic buffers with cobaltic hexaammine (III) (Co(NH3)63+). We find that neither the critical concentration of Co(NH3)63+ nor the hydrodynamic radii of the resulting condensates vary significantly in the buffer systems studied here despite the prediction that DNA condensation should occur at significantly lower Co(NH3)63+ concentrations in Tris-borate buffers. Assuming a persistence length behavior similar to B-DNA in the presence of multivalent cations, a decrease in the attractive counterion correlation pressure decay length in Tris-borate buffers does not account for our observations. It is possible that the binding of multivalent cations to DNA may hinder borate association with the DNA double helix.

Interactions of Spermidine and Spermine with Genomic B DNA of Differing GC/AT Content: Investigation by Raman Spectroscopy
H. Deng, V. A. Bloomfield, J. M. Benevides and G. J. Thomas, Jr. (2000) Nucleic Acids Res. 28: 3379-3385. pdf

Four genomic DNAs of differing GC/AT content (M. luteus,72% GC; E. coli, 50% GC; calf thymus, 42% GC; C. perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines, spermidine {[H3N(CH2)3NH2(CH2)4NH3]3+} and spermine {[(CH2)4(NH2(CH2)3NH3)2]4+}. In solutions containing polyamine at either 1, 5 or 60 mM and genomic DNA at 60 mM phosphate (~20 mg DNA/mL), only Raman bands associated with localized vibrational modes of the DNA phosphates exhibit large spectral perturbations, indicating that phosphates of the B-DNA backbone are the primary targets of both spermidine and spermine interactions. This finding supports previously proposed models in which polyamine-induced DNA condensation is driven by nonspecific electrostatic bonding. However, the Raman spectra indicate that major-groove sites (guanine N7 and thymine C5H3 groups) of genomic B DNA are also affected, though less significantly than the phosphates, by polyamine interactions. The altogether modest dependence of polyamine binding on base composition in genomic DNA suggests that sequence context plays only a secondary role in polyamine recognition. Importantly, the Raman spectra demonstrate that binding of polyamines to genomic DNA has negligible effect on the B-form secondary structure. Each polyamine-bound genomic DNA retains the B conformation when highly condensed (fully precipitated from solution). The capability of spermidine or spermine to bind and condense genomic DNA without disrupting the native B structure is considered biologically significant and discussed in relation to previously proposed models.

Excluded volume in solvation: sensitivity of scaled-particle theory to solvent size
Tang, K.E.S. and Bloomfield, V.A. (2000), Biophys. J., 79: 2222-2234. pdf

Changes in solvent environment greatly affect macromolecular structure and stability. To understand the physics behind solvation, the free energy of solute transfer from one environment to another, DGtr, is often split into more fundamental constituents---an excluded-volume portion, DGtrev, commonly calculated with scaled-particle theory (SPT), and a soft-interaction part. SPT requires the hard-sphere radii of the solvent and solute molecules as input parameters. However, for any particular molecular species, different experiments give different radii. Here, we use SPT to estimate the uncertainty in DGtrev due to the uncertainty in solvent radii. For the transfer of amino-acid solutes from water to water-osmolyte solutions, we show that the uncertainties of DGtrev values are in the range of several kcals/mol---as large as the values themselves; sometimes even the sign of DGtrev is unclear. In comparison, experimentally measured DGtrev values are an order of magnitude smaller. Estimating the excluded-volume portion of DGtr using a model which represents the solvent molecules crudely by a few parameters is problematic. Lastly, we discuss why DGtrev is so sensitive to solvent size, and show that this sensitivity will likely exist for any excluded-volume calculation, regardless of model.

Last updated 4/7/02

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