Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching.

The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid chaperone that facilitates the rearrangement of nucleic acids into conformations containing the maximum number of complementary base pairs. We use an optical tweezers instrument to stretch single DNA molecules from the helix to coil state at room temperature in the presence of NC and a mutant form (SSHS NC) that lacks the two zinc finger structures present in NC. Although both NC and SSHS NC facilitate annealing of complementary strands through electrostatic attraction, only NC destabilizes the helical form of DNA and reduces the cooperativity of the helix-coil transition. In particular, we find that the helix-coil transition free energy at room temperature is significantly reduced in the presence of NC. Thus, upon NC binding, it is likely that thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations to find the lowest energy state. The reduced cooperativity allows these fluctuations to occur in the middle of complex double-stranded structures. The reduced stability and cooperativity, coupled with the electrostatic attraction generated by the high charge density of NC, is responsible for the nucleic acid chaperone activity of this protein.

Entropy and heat capacity of DNA melting from temperature dependence of single molecule stretching.

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have measured the force at which this transition occurs as a function of temperature. To do this, single molecules of DNA were captured between two polystyrene beads in an optical tweezers apparatus. As the temperature of the solution surrounding a captured molecule was increased from 11 degrees C to 52 degrees C in 500 mM NaCl, the overstretching transition force decreased from 69 pN to 50 pN. This reduction is attributed to a decrease in the stability of the DNA double helix with increasing temperature. These results quantitatively agree with a model that asserts that DNA melting occurs during the overstretching transition. With this model, the data may be analyzed to obtain the change in the melting entropy DeltaS of DNA with temperature. The observed nonlinear temperature dependence of DeltaS is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be DeltaC(p) = 60 +/- 10 cal/mol K bp, in agreement with calorimetric measurements.

Effect of pH on the overstretching transition of double-stranded DNA: evidence of force-induced DNA melting.

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have investigated the source of this transition by altering helix stability with solution pH. As solution pH was increased from pH 6.0 to pH 10.6 in 250 mM NaCl, the overstretching transition force decreased from 67.0 +/- 0.8 pN to 56.2 +/- 0.8 pN, whereas the transition width remained nearly constant. As the pH was lowered from pH 6.0 to pH 3.1, the overstretching force decreased from 67.0 +/- 0.8 pN to 47.0 +/- 1.0 pN, but the transition width increased from 3.0 +/- 0.6 pN to 16.0 +/- 3 pN. These results quantitatively agree with a model that asserts that DNA strand dissociation, or melting, occurs during the overstretching transition.

Crowding effects on EcoRV kinetics and binding.

The cytosol of the cell contains high concentrations of small and large macromolecules, but experimental data are often obtained in dilute solutions that do not reflect in vivo conditions. We have studied the crowding effect that large macromolecules have on EcoRV cleavage by adding high-molecular-weight Ficoll 70 to reaction solutions. Results indicate that Ficoll has surprisingly little effect on overall EcoRV reaction velocity because of offsetting increases in V(max) and K(m), and stronger nonspecific binding. The changes in measured parameters can largely be attributed to the excluded volume effects on reactant activities and the slowing of protein diffusion. Covolume reduction upon binding appears to reinforce nonspecific binding strength, and k(cat) appears to be slowed by stronger nonspecific binding, which slows product release. The data also suggest that effective Ficoll particle volume decreases as its concentration increases above a few weight percent, which may be due to Ficoll interpenetration or compression.

Buffer effects on EcoRV kinetics as measured by fluorescent staining and digital imaging of plasmid cleavage.

We have developed a protocol to quantify polymer DNA cleavage which replaces the traditional radiolabeling and scintillation counting with fluorescent staining and digital imaging. This procedure offers high sensitivity, speed, and convenience, while avoiding waste and error associated with traditional 32P radiolabeling. This protocol was used to measure cleavage of pBR322 plasmid DNA by EcoRV, a type II restriction enzyme. EcoRV was found to exhibit an order of magnitude difference in binding in two apparently similar buffers used in previous investigations. To determine the origin of this effect, we measured reaction kinetics in buffers of different chemical nature and concentration: Tris, bis-Tris propane, Tes, Hepes, and cacodylate. We found that buffer concentration and identity had significant effects on EcoRV reaction velocity through large changes in specific binding and nonspecific binding (reflected in the Michaelis constant Km and the dissociation constant for nonspecific binding Kns). There were only small changes in Vmax. The source of the buffer effect is the protonated amines common to many pH buffers. These buffer cations likely act as counterions screening DNA phosphates, where both the protonated buffer structure and concentration affect enzyme binding strength. It appears that by choosing anionic buffers or zwitterionic buffers with a buried positive charge, buffer influence on the protein binding to DNA can be largely eliminated.

Osmotic pressure effects on EcoRV cleavage and binding.

Investigations of DNA binding proteins frequently measure pH and salt dependence, but relatively few studies measure protein binding in high concentrations of small molecules often found in vivo. We have measured kinetics of the restriction enzyme EcoRV in concentrated solutions of three small cosolvents that produce osmotic pressures from 0.25 to 2.5 mol/kg (6 to 62 atm or water activity of 0.995 to 0.956). We have correlated DNA cleavage and binding parameters with four solution parameters (dielectric constant, viscosity, water concentration, and water activity). We found that the responses of maximum velocity (Vmax) and the dissociation constant for nonspecific binding (Kd,ns) best correlate with water activity. The Michaelis constant (Km) correlates with both water activity and solution viscosity, the latter due to the highly dilute reactant concentrations, which make enzyme-substrate combination diffusion limited. Dielectric constant does not influence any of the kinetic parameters, which is consistent with a view that protein and DNA are preferentially hydrated, and excluded solutes cannot affect the local dielectric constant.

DNA condensation by cobalt hexaammine (III) in alcohol-water mixtures: dielectric constant and other solvent effects.

DNA molecules condense into compact structures in the presence of a critical concentration of multivalent cations. To probe the contribution of electrostatic forces to condensation, we used mixtures of water with methanol (MeOH), ethanol (EtOH), and isopropanol (iPrOH) to vary the dielectric constant epsilon from 80 to 50. The condensation of pUC18 plasmids by hexaammine cobalt (III), Co(NH3)(3+)6, was monitored by total intensity and dynamic light scattering, electron microscopy, and CD. The total scattering intensity increased as epsilon went from 80 to 70, and the decreased as epsilon decreased further. Ultraviolet spectrophotometry confirmed that the loss of intensity at low epsilon was not due to the particles' settling out of solution. The rate as well as the extent of condensation increased as epsilon was lowered from 80 to 70, and also depended on the species of alcohol (MeOH < EtOH < iPrOH). The hydrodynamic radii RH of the particles, however, remained roughly the same at 300-350 A and was independent of the species of alcohol. RH increased below epsilon = 70. The critical concentration of Co(NH3)6(3+) required to induce DNA condensation decreased from 21 microM to about 16 microM as the dielectric constant decreased from 80 to 70, and decreased moderately with the nonpolarity of the alcohol. The fraction of DNA charge neutralized at the onset of DNA condensation was calculated by a modification of Manning's two-variable counterion condensation theory to be 0.90 +/- 0.01, independent of epsilon. By electron microscopy we observed that the condensed particles changed from about 93% toroids at epsilon = 80 to 89% rods at epsilon = 70 and 98% rods at epsilon = 65. At epsilon lower than 65, DNA collapsed into a network of multistranded fibers. The morphology of condensed DNA particles, whether toroids, rods, or fibers, was independent of the alcohol species. CD spectra in ethanol-water mixtures indicated that both closed circular and linearized plasmids were in the B conformation when condensed with Co(NH3)6(3+) at epsilon > or = 70, although the closed circular molecules exhibited a weak psi-DNA spectrum. A transition from the B to A form took place between epsilon = 70 and 60, well above the normal dielectric constant of epsilon = 40 for this transition, indicating that ethanol and Co(NH3)6(3+) synergistically promote the B-A transition. We interpret these results to mean that alcohols have both electrostatic and structural effects on DNA, leading to three regimes of condensation. At the lowest alcohol concentrations the B conformation is stable and condensation is relatively slow, allowing time for the packing adjustments necessary to form toroids.(ABSTRACT TRUNCATED AT 400 WORDS)