Abstracts of 2001 Publications


The effect of pH on the overstretching transition of dsDNA: Evidence of force-induced DNA melting
Williams, M.C., Wenner, J.R., Rouzina, I. and Bloomfield, V.A. (2001) Biophys. J. 80: 874-881 pdf

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 pN to 56 pN, while 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 pN to 47 pN, but the transition width increased from 3 pN to 16 pN. These results quantitatively agree with a model that asserts that DNA strand dissociation, or melting, occurs during the overstretching transition.

Force-induced melting of the DNA double helix. 1. Thermodynamic analysis
Rouzina, I. and Bloomfield, V.A. (2001) Biophys. J. 80: 882-893 pdf

The highly cooperative elongation of a single B-DNA molecule to almost twice its contour length upon application of a stretching force is interpreted as force-induced DNA melting. This interpretation is based on the similarity between experimental and calculated stretching profiles, when the force-dependent free energy of melting is obtained directly from the experimental force vs extension curves of double- and single-stranded DNA. The high cooperativity of the overstretching transition is consistent with a melting interpretation. The ability of nicked DNA to withstand forces greater than that at the transition midpoint is explained as a result of the one-dimensional nature of the melting transition, which leads to alternating zones of melted and unmelted DNA even substantially above the melting midpoint. We discuss the relationship between force-induced melting and the B to S transition suggested by other authors. The recently measured effect on T7 DNA polymerase activity of the force applied to a ssDNA template is interpreted in terms of preferential stabilization of dsDNA by weak forces of about 7 pN.

Force-induced melting of the DNA double helix. 2. Effect of solution conditions
Rouzina, I. and Bloomfield, V.A. (2001) Biophys. J. 80: 894-900 pdf

In this paper we consider the implications of the general theory developed in the accompanying paper, to interpret experiments on DNA overstretching that involve variables such as solution temperature, pH, and ionic strength. We find the DNA helix-coil phase boundary in the force-temperature space. At temperatures significantly below the regular (zero force) DNA melting temperature, the overstretching force f_ov is predicted to decrease nearly linearly with temperature. We calculate the slope of this dependence as a function of entropy and heat capacity changes upon DNA melting. Fitting of the experimental f_ov(T) dependence allows determination of both of these quantities in very good agreement with their calorimetric values. At temperatures slightly above the regular DNA melting temperature we predict stabilization of dsDNA by moderate forces, and destabilization by higher forces. Thus the DNA stretching curves, f(b), should exhibit two rather than one overstretching transitions: from ss to ds and then back at the higher force. We also predict that any change in DNA solution conditions that affects its melting temperature should have a similar effect on DNA overstretching force. This result is used to calculate the dependence of DNA overstretching force on solution pH, f_ov(pH), from the known dependence of DNA melting temperature on pH. The calculated f_ov(pH) is in excellent agreement with its experimental determination {Williams et al, 2001}. Finally, we quantitatively explain the measured dependence of DNA overstretching force on solution ionic strength for crosslinked and noncrosslinked DNA. The much stronger salt dependence of f_ov} in noncrosslinked DNA results from its lower linear charge density in the melted state, compared to crosslinked or double-stranded overstretched S-DNA.

Entropy and Heat Capacity of DNA Melting from Temperature Dependence of Single Molecule Stretching
Williams, M.C., Wenner, J..R., Rouzina, I. and Bloomfield, V.A. (2001) Biophys. J., 80: 1932-1939 pdf

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 C to 52 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 Delta S of DNA with temperature. The observed nonlinear temperature dependence of Delta S is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be Delta Cp = 60 +/- 10 cal/mol-K-bp, in agreement with calorimetric measurements.

Role of zinc fingers in nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching
M.C. Williams, J.R. Wenner, I. Rouzina, R.J. Gorelick, K. Musier-Forsyth, and V.A. Bloomfield (2001) Proc. Natl. Acad. Sci. 98: 6121-6126. pdf

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-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, thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order 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.

Hexamminecobalt(III)-induced condensation of calf thymus DNA. Circular dichroism, ultrasonic and density measurements
B.I. Kankia, V. Buckin and V.A. Bloomfield (2001) Nucleic Acids Res., 29: 2795-2801. pdf

The interaction of hexamminecobalt(III), Co(NH3)63+, with 160 and 3000-8000 bp length calf thymus DNA has been investigated by circular dichroism, acoustic and densimetric techniques. The acoustic titration curves of 160 bp DNA revealed three stages of interaction: (i) Co(NH3)63+ binding up to the molar ration [Co(NH3)63+]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH3)63+]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH3)63+]/[P] = 0.3. In the case of 3000-8000 DNA only two processes were observed: (i) binding up to [Co(NH3)63+]/[P] = 0.3; and (ii) precipitation after this point. In agreement with earlier observations, long DNA aggregates without changes in its B-form circular dichroism spectrum, while short DNA demonstrates a positive B -> Psi transition after [Co(NH3)63+]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH3)63+ binding on volume and compressibility have been obtained. The binding of Co(NH3)63+ to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH3)63+: Delta(V) = 9 cc/mol and Delta(kappa) = 33 x 10(-4) cc/mol-bar. The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH3)63+ and the atomic groups of DNA. This extent of water displacement would be consistent with the formation of two direct, hydrogen bonded contacts between the cation and the phosphates of DNA.

Thermodynamics of the Hydrophobic Effect. I. Coupling of Aggregation and pKa Shifts in Solutions of Aliphatic Amines
D. Matulis and V.A. Bloomfield (2001) Biophy. Chem., 93: 37-51. pdf

Long aliphatic hydrocarbon chains aggregate in aqueous solution due to the hydrophobic effect, forming structures such as micelles and membranes, while amino groups titrate at basic pH. These two biologically important behaviors are linked in alkylamines, in which the pKa of the amino group is shifted downward by aggregation. In this paper we study the thermodynamics of these coupled processes, following aggregation by observing alkylamine pH titration behavior. The magnitude of the shift depended on the aliphatic chain length and on the concentration of alkylamine: longer chains and higher concentrations lowered the pKa to a greater extent. Gibbs free energies of protonation and aggregation were calculated from the pKa shifts. Enthalpies, entropies, and heat capacities were estimated by van't Hoff analysis from the pKa shift dependencies on temperature. However, the results were less precise than the calorimetrically measured values, as described in the following article. A model to calculate titration curves, pKa shifts, and aggregation of uncharged alkylamines as a function of aliphatic chain length, concentration, and temperature is presented.

Thermodynamics of the Hydrophobic Effect. II. Calorimetric Measurement of Enthalpy, Entropy, and Heat Capacity of Aggregation of Alkylamines and Long Aliphatic Chains
D. Matulis and V.A. Bloomfield (2001) Biophys. Chem., 93: 53-65. pdf

The thermodynamics of long aliphatic chain alkylamine aggregation in aqueous solution was studied by isothermal titration calorimetry (ITC). Protonated alkylammonium cations with linear aliphatic chains of 10 to 14 carbon atoms were fully soluble in aqueous solution at the beginning of titration, but practically insoluble after deprotonation by titrating with sodium hydroxide. The alkylamines aggregated and precipitated during the reaction, enabling direct measurement of the enthalpy of aggregation. The enthalpy of aggregation became increasingly exothermic upon increasing the chain length. Hydrophobic aggregation was enthalpy-driven and entropy-opposed for alkylamines with 12 to 14 carbon atoms at room temperature. Direct observation of hydrophobic aggregation by ITC at constant temperature and pressure provided more accurate thermodynamic parameters than obtainable from van't Hoff analysis. Aggregation into liquid or solid phases could be distinguished by ITC, but not by van't Hoff analysis of alkylamine solubility data.

Thermodynamics of the Hydrophobic Effect. III. Condensation and Aggregation of Alkanes, Alcohols, and Alkylamines
D. Matulis (2001) Biophy. Chem., 93: 67-82. pdf

Knowledge of the energetics of the low solubility of nonpolar compounds in water is critical for the understanding of such phenomena as protein folding and biomembrane formation. Solubility in water can be considered as one leg of the three-part thermodynamic cycle - vaporization from the pure liquid, hydration of the vapor in aqueous solution, and aggregation of the substance back into initial pure form as an immiscible phase. Previous studies on the model compounds n-alkanes, 1-alcohols, and 1-aminoalkanes have noted that the thermodynamic parameters (Gibbs free energy DG, enthalpy DH, entropy, DS, and heat capacity, DCp) associated with these three processes are generally linear functions of the number of carbons in the alkyl chains. Here we assess the accuracy and limitations of the assumption of additivity of CH2 group contributions to the thermodynamic parameters for vaporization, hydration, and aggregation. Processes of condensation from pure gas to liquid and aqueous solution to aggregate are compared. Hydroxy, amino, and methyl headgroup contributions are estimated, liquid and solid aggregates are distinguished. Most data in the literature were obtained for compounds with short aliphatic hydrocarbon tails. Here we emphasize long aliphatic chain behavior and include our recent experimental data on long chain alkylamine aggregation in aqueous solution obtained by titration calorimetry and van't Hoff analysis. Contrary to what is observed for short compounds, long aliphatic compound aggregation has a large exothermic enthalpy and negative entropy.

Last updated 4/7/02

Recent Publications
Home Page