Abstracts of 1999 Publications


Matulis, D., Baumann, C.G., Bloomfield, V.A. and Lovrien, R. (1999) Biopolymers 49: 451-458. 1-Anilino-8-Naphthalene Sulfonate as a Protein Conformational Tightening Agent.pdf

The 1-anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins through its nonpolar anilinonaphthalene group. Such binding is followed by an increase of ANS fluorescence intensity, similar to that occurring when ANS is dissolved in organic solvents. It is generally assumed that neither the negative sulfonate charge on the ANS, nor charges on the protein, participate significantly in ANS-protein interaction. However, equilibrium dialysis and titration calorimetry have demonstrated that the majority of ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups of the proteins (Matulis and Lovrien, 1998). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin (BSA) and g-globulin, starting from their acid expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. ANS anion binding and molecular compaction are accompanied by the Scatchard-Black effect, an upward shift in pH as the protein charge density becomes more negative and OH- is released into solution. These results show that, over a large range of binding to BSA and g-globulin in acidic solutions, ANS is not a reliable reporter of the protein molecular structure that existed before ANS binding. Instead, it reports a conformationally tightened state produced by the interplay of hydrophobic and ionic characters of both protein and ligand.


Wenner, J.R. and Bloomfield, V.A. (1999) Anal. Biochem. 268: 201-212. Buffer Effects on EcoRV Cleavage and Binding to Plasmid DNA. pdf

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.


Deng, H. and Bloomfield, V.A. (1999) Biophys. J., 77: 1556-1561. Structural effects of cobalt-amine compounds on DNA condensation. pdf

Light scattering and electron microscopy have been used to investigate structural effects of the trivalent complexes hexaammine cobalt (III) chloride (Cohex), tris(ethylenediamine) cobalt(III) chloride (Coen), and cobalt(III) sepulchrate chloride (Cosep) on DNA condensation. These cobalt-amine compounds have similar ligand coordination geometry, but differ slightly in size. Their hydrophobicity is in the order Cosep $>$ Coen $>$ Cohex according to the numbers of methylene groups in these ligands. All of these compounds effectively precipitate DNA at high concentrations; but despite a lower surface charge density, Cosep condenses DNA twice as effectively as Coen or Cohex. UV and CD measurements on the supernatants of cobalt-amine/DNA solutions reveal a preferential binding of $\Delta$-Coen over $\Lambda$-Coen to the precipitated DNA, but there is no chiral selectivity for Cosep. Competition experiments show that the binding strengths of these three cobalt-amine compounds to aggregated DNA are comparable. A charge neutralization of 88-90\% is required for DNA condensation. Our data indicate that (1) electrostatic interaction is the main driving force for binding of multivalent cations to DNA; (2) DNA condensation is dependent on the structure of the condensing agent; and (3) the hydration pattern or polarization of water molecules on the surface of condensing agents plays an important role in DNA condensation and chiral recognition.


Deng, H., Bloomfield, V.A., Benevides, J. and Thomas, G.J., Jr. (1999) Biopolymers, 50: 656-666. Dependence of the Raman signature of genomic B DNA on nucleotide base sequence.pdf

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT)×poly(dA-dT), 0% G+C; C. perfringens DNA, 27% G+C; calf thymus DNA, 42% G+C; E. coli DNA, 50% G+C; M. luteus DNA, 72% G+C; poly(dG-dC)×poly(dG-dC), 100% G+C], have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases - particularly of A and T - exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.


Rouzina, I. and V.A. Bloomfield (1999), "Heat Capacity Effects on the Melting of DNA. 1. General Aspects", Biophys. J. 77: 3242-3251. pdf

In this paper we analyze published data on \DelH\ and \DelS\ values for the DNA melting transition under various conditions. We show that there is a significant heat capacity increase \DelCp\ associated with DNA melting, in the range 40--100 cal/mol K per base pair. This is larger than the transition entropy per base pair $\DelS^{0} \approx$ 25 cal/mol K. The ratio of $\DelCp/ \DelS^{0}$ determines the importance of heat capacity effects on melting. For DNA this ratio is 2--4, larger than for many proteins. We discuss how \DelCp\ values can be extracted from experimental data on the dependence of \DelH\ and \DelS\ on melting temperature \Tm. We consider studies of DNA melting as a function of ionic strength, and show that while polyelectrolyte theory provides a good description of the dependence of \Tm\ on salt, electrostatics alone cannot explain the accompanying strong variation of \DelH\ and \DelS. While \Tm\ is only weakly affected by \DelCp, its dependence on one parameter (e.g. salt) as a function of another (e.g. DNA composition) is determined by \DelCp. We show how this accounts for the stronger stabilization of AT relative to GC base pairs with increasing ionic strength. We analyze the source of discrepancies in \DelH\ as determined by calorimetry and van't Hoff analysis, and discuss ways of analyzing data that yield valid van't Hoff \DelH. Finally, we define a standard state for DNA melting, the temperature at which thermal contributions to \DelH\ and \DelS\ vanish, by analyzing experimental data over a broad range of stabilities.


Rouzina, I. and V.A. Bloomfield (1999), "Heat Capacity Effects on the Melting of DNA. 2. Analysis of Nearest Neighbor Base Pair Effects", Biophys. J. 77: 3252-3255. pdf

The stability of a DNA double helix of any particular sequence is conventionally estimated as the average of the stabilities of the ten different nearest neighbor (NN) base pair doublets that it contains. Therefore, much effort has been devoted to experimental characterization and tabulation of the enthalpy, entropy and free energy of melting for each of the NN doublets. While data from different research groups generally agree for the NN free energies and melting temperatures, there are major disagreements for the enthalpies and entropies. The largest differences are between the parameters obtained on oligomeric relative to polymeric DNA. This disagreement interferes with the practical application of NN thermodynamic parameters. It also raises doubts regarding several fundamental assumptions about DNA melting, such as absence of longer range interactions, length dependence of DNA melting parameters per base pair, applicability of polyelectrolyte theory for describing salt effects on oligomers, and purely enthalpic difference between NN doublets. Here we show that if one takes into account the significant heat capacity increase associated with DNA melting, all of the above assumptions are self-consistently reconciled with experiment.


Wenner, J.R. and V.A. Bloomfield (1999), "Crowding Effects on EcoRV Kinetics and Binding", Biophys. J., 77: 3234-3241.pdf

The cytosol of the cell contains high concentrations of small and large macromolecules, but experimental data are often obtained in dilute solutions which do not reflect in vivo conditions. The previous communication has shown that small cosolvents change EcoRV cleavage parameters by lowering water activity and slowing protein diffusion. In this work, we study the effects of large macromolecules 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 due to offsetting increases in Vmax and Km, 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 kcat 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.


Osmotic Pressure Effects on EcoRV Cleavage and Binding, Wenner, J.R. and V.A. Bloomfield (1999), J. Biomol. Struct. Dynam.17: 461-471.

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 a water activity of 0.995 to 0.956). We have correlated cleavage and binding parameters with four solution parameters (dielectric constant, viscosity, water concentration, and water activity). We found that the response of maximum velocity (Vmax) and the dissociation constant for nonspecific binding (Kns) best correlates 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.

In studies of restriction endonucleases, there has been debate on whether EcoRV and EcoRI are fundamentally similar or different. The EcoRV cleavage and nonspecific binding results determined here are similar to those obtained for EcoRI, suggesting that the enzymes have more in common than may have been inferred from a previous osmotic pressure study (Robinson & Sligar, 1995).


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