Scanning tunneling microscopy of nucleic acids.

The scanning tunneling microscope (STM) has been used to measure properties of poly(rA).poly(rU) and DNA, such as helical pitch, half-period oscillations that were interpreted as the alternation between the major and minor grooves, and interhelical spacing. Average pitches were measured by two-dimensional Fourier transforms and by topographic profiles along the fiber axes. Values were typically 7 percent less than standard dimensions of A-form RNA and B-form DNA fibers. This result is compatible with the mild dehydration that occurred under air-drying conditions. More extensive dehydration typically led to 19 percent shrinkage. Analysis of specific regions allowed local variations in helical pitch as small as 1 angstrom to be detected, thus demonstrating that the STM can visualize functionally significant modulations of nucleic acid structure.

DNA condensation.

Recent progress in our understanding of DNA condensation includes the observation of the collapse of single DNA molecules, greater insights into the intermolecular forces driving condensation, the recognition of helix-structure perturbation in condensed DNA, and the increasing recognition of the likely biological consequences of condensation. DNA condensed with cationic liposomes is an efficient agent for the transfection of eukaryotic cells, with considerable potential interest for gene therapy.

Hexamminecobalt(III)-induced condensation of calf thymus DNA: circular dichroism and hydration measurements.

The interaction of hexamminecobalt(III), Co(NH(3))(6)(3+), 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(NH(3))(6)(3+) binding up to the molar ratio [Co(NH(3))(6)(3+)]/[P] = 0.25, prior to DNA condensation; (ii) a condensation process between [Co(NH(3))(6)(3+)]/[P] = 0.25 and 0.30; and (iii) precipitation after [Co(NH(3))(6)(3+)]/[P] = 0.3. In the case of 3000-8000 bp DNA only two processes were observed: (i) binding up to [Co(NH(3))(6)(3+)]/[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(NH(3))(6)(3+)]/[P] = 0.25. From ultrasonic and densimetric measurements the effects of Co(NH(3))(6)(3+) binding on volume and compressibility have been obtained. The binding of Co(NH(3))(6)(3+) to both short and long DNA is characterized by similar changes in volume and compressibility calculated per mole Co(NH(3))(6)(3+): DeltaV = 9 cm(3) mol(-1) and Deltakappa = 33 x 10(-4) cm(3) mol(-1) bar(-1). The positive sign of the parameters indicates dehydration, i.e. water release from Co(NH(3))(6)(3+) 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.

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy.

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine ([H(3)N(CH(2))(3)NH(2)(CH(2))(4)NH(3)](3+)) and spermine ([(CH(2))(4)(NH(2)(CH(2))(3)NH(3))(2)](4+)). In solutions containing 60 mM DNA phosphate (approximately 20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H(3)) are less affected than phosphates by polyamine-DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.

Effect of chymosin action on the hydrodynamic diameter of casein micelles.

Quasi-elastic light scattering shows an initial decrease of about 5 nm in the hydrodynamic radius of casein micelles after adding chymosin, assuming the decrease to be equal for all micelles. This is consistent with the hypothesis that casein micelles have a hairy outer layer that is partly made up of the caseino-macropeptide part of kappa casein.

The homo-dimeric form of ADP-ribosyl cyclase in solution.

ADP-ribosyl cyclase is a multi-functional enzyme that catalyzes the formation of two Ca2+ signaling molecules, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). X-ray crystallography of three different crystal forms shows that it is a non-covalent dimer. Chemical cross-linking and dynamic light scattering were used in this study to determine if the cyclase is also a non-covalent dimer in solution. Treatment of the cyclase in dilute solution (0.05 mg/ml) with dimethylsuberimidate resulted in complete conversion to a species with molecular weight about twice that of the monomeric cyclase. Prolonged cross-linking of the cyclase at four times higher concentration produced also only the covalently linked dimers and no multimer formation was observed. The cross-linked dimer retained full enzymatic activity and readily catalyzed the formation of cADPR from NAD, NAADP from NADP, cyclic ADP-ribose phosphate from NADP, and cyclic GDP-ribose from nicotinamide guanine dinucleotide. Analysis of the autocorrelation functions obtained from dynamic light scattering measurements indicated the cyclase solution (2 mg/ml) was composed of a single molecular species and its diffusion coefficient was measured to be 7. 4x10-7 cm2/s. Computer modeling using the crystallographic dimensions of the non-covalent cyclase dimer, a donut shaped molecule with a central cavity and overall dimensions of 7x6x3 nm, gave a value for the diffusion coefficient essentially the same as that measured. These results indicate the cyclase is a non-covalent dimer in solution.

Thermodynamics of DNA binding and condensation: isothermal titration calorimetry and electrostatic mechanism.

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 (TDeltaS approximately +10 kcal/mol cation) and enthalpically opposed (DeltaH approximately +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.

Scanning tunnelling microscopy in biotechnology.

The scanning tunnelling microscope (STM) is capable of atomic resolution of highly conductive materials. Whether biological molecules can be visualized to the same extent remains an open question, but remarkable progress in the past year confirms the possibility of seeing the fine structure of nucleic acids, proteins, membranes and viruses, and provides evidence that their dynamic interactions can be monitored under conditions approximating to those of the native environment.

Competitive electrostatic binding of charged ligands to polyelectrolytes: practical approach using the non-linear Poisson-Boltzmann equation.

We have developed a practical analytical treatment of the non-linear Poisson-Boltzmann (P-B) equation to characterize the strong but non-specific binding of charged ligands to DNA and other highly charged macromolecules. These reactions are notable for their strong salt dependence and anti-cooperativity, features which the theory fully explains. We summarize analytical results for concentration profiles and ion binding in various regimes of surface curvature and ionic strength, and show how counterion size and charge distribution may influence competitive binding. We present several practical applications of the formalism, showing how to estimate the ligand concentration needed to effectively compete with a given buffer salt, and how to calculate the amounts of counterion species bound at various distances from the DNA surface under given bulk solution conditions. We cast our results into the form of a Scatchard binding isotherm, showing how the apparent binding constant K(obs) and S = -dlog K (obs )d log[M (+)] can be predicted from the basic theory. Anti-cooperativity arises naturally without steric repulsion, and binding curves can be fitted with K(obs) and effective charge as the only free parameters. We extend the analytical P-B analysis to an arbitrary number of counterion species, and apply the results to fit and predict three-ion competition data.

Determination of polymer size distribution by combination of quasielastic light scattering and band transport: evaluation of the effect of diffusion.

In this paper we report a computer simulation study of the effect of diffusion on the size distribution obtained by combining light scattering with isokinetic band sedimentation or electrophoresis. We find that, under typical experimental conditions, the method yields reasonably accurate size distributions for samples of particles greater than 10 nm radius. However, caution should be exercised in interpreting the results for smaller particles, for which the distortion due to diffusion can be considerable.

Thermodynamics of the hydrophobic effect. I. Coupling of aggregation and pK(a) shifts in solutions of aliphatic amines.

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 pK(a) 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 pK(a) to a greater extent. Gibbs free energies of protonation and aggregation were calculated from the pK(a) shifts. Enthalpies, entropies, and heat capacities were estimated by van't Hoff analysis from the pK(a) 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, pK(a) 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.

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 10-14 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 12-14 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.

Brownian dynamics simulation of probe diffusion in DNA: effects of probe size, charge and DNA concentration.

We have used Brownian dynamics simulation to study probe diffusion in solutions of short chain DNA using our previously developed simulation algorithm. We have examined the effect of probe size, charge, and DNA concentration on the probe diffusion coefficient, with the aim of gaining insight into the diffusion of proteins in a concentrated DNA environment. In these simulations, DNA was modeled as a worm-like chain of hydrodynamically equivalent spherical frictional elements while probe particles were modeled as spheres of given charge and hydrodynamic radius. The simulations allowed for both short range Lennard-Jones interactions and long ranged electrostatic interactions between charged particles. For uncharged systems, we find that the effects of probe size and DNA concentration on the probe diffusion coefficient are consistent with excluded volume models and we interpret our results in terms of both empirical scaling laws and the predictions of scaled particle theory. For charged systems, we observe that the effects of probe size and charge are most pronounced for the smallest probes and interpret the results in terms of the probe charge density. For an ionic strength of 0.1 M we find that, below a critical probe surface charge density, the probe diffusion coefficient is largely independent of probe charge and only weakly dependent on the DNA charge. These effects are discussed in terms of the interactions between the probe and the DNA matrix and are interpreted in terms of both the underlying physics of transport in concentrated solutions and the assumptions of the simulation model.

Polyelectrolyte effects in DNA condensation by polyamines.

The conditions required for the counterion induced collapse of T7 bacteriophage DNA are briefly reviewed. Using Manning's counterion condensation theory we calculate a striking unity among collapse conditions: collapse occurs when from 89% to 90% of the DNA phosphate charges are neutralized by condensed counterions. The forces involved in collapsed DNA are investigated with emphasis on electrostatic repulsion. It is concluded that polyelectrolyte repulsion is the dominant force opposing collapse. Comparison of the results or polyelectrolyte repulsion calculation made using numerical methods and the Poisson-Boltzmann equation, with values of the attractive energies due to London dispersion interactions, leads to the conclusion that dispersion forces are probably large enough to cause collapse when the repulsions have been reduced by the presence of multivalent counterions.

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.