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V A Bloomfield

Publications and source records attributed to V A Bloomfield.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Buffers↗
PubMed1999

1-anilino-8-naphthalene sulfonate as a protein conformational tightening agent.

1-Anilino-8-naphthalene sulfonate (ANS) anion is conventionally considered to bind to preexisting hydrophobic (nonpolar) surfaces of proteins, primarily through its nonpolar anilino-naphthalene group. Such binding is followed by an increase in 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, titration calorimetry has demonstrated that most ANS binding to a number of proteins occurs through electrostatic forces, in which ion pairs are formed between ANS sulfonate groups and cationic groups on the proteins (D. Matulis and R. E. Lovrien, Biophys. J., 1998, Vol. 74, pp. 1-8). Here we show by viscometry and diffusion coefficient measurements that bovine serum albumin and gamma-globulin, starting from their acid-expanded, most hydrated conformations, undergo extensive molecular compaction upon ANS binding. As the cationic protein binds negatively charged ANS anion it also takes up positively charged protons from water to compensate the effect of the negative charge, and leaves the free hydroxide anions in solution thus shifting pH upward (the Scatchard-Black effect). These results indicate that ANS is not always a definitive reporter of protein molecular conformation that existed before ANS binding. Instead, ANS reports on a conformationally tightened state produced by the interplay of ionic and hydrophobic characters of both protein and ligand.

Anilino Naphthalenesulfonates↗
PubMed1999

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

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; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), 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.

Animals↗
PubMed1999

Structural effects of cobalt-amine compounds on DNA condensation.

Light scattering and electron microscopy have been used to investigate the 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 geometries 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 of 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.

Amines↗
PubMed1999

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.

Biophysical Phenomena↗
PubMed1999

Heat capacity effects on the melting of DNA. 1. General aspects.

In this paper we analyze published data on DeltaH and DeltaS values for the DNA melting transition under various conditions. We show that there is a significant heat capacity increase DeltaC(p) associated with DNA melting, in the range of 40-100 cal/mol K per base pair. This is larger than the transition entropy per base pair, DeltaS(0) approximately 25 cal/mol K. The ratio of DeltaC(p)/DeltaS(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 DeltaC(p) values can be extracted from experimental data on the dependence of DeltaH and DeltaS on the melting temperature T(m). 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 T(m) on salt, electrostatics alone cannot explain the accompanying strong variation of DeltaH and DeltaS. While T(m) is only weakly affected by DeltaC(p), its dependence on one parameter (e.g., salt) as a function of another (e.g., DNA composition) is determined by DeltaC(p). 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 DeltaH as determined by calorimetry and van't Hoff analysis and discuss ways of analyzing data that yield valid van't Hoff DeltaH. Finally, we define a standard state for DNA melting, the temperature at which thermal contributions to DeltaH and DeltaS vanish, by analyzing experimental data over a broad range of stabilities.

Base Pairing↗
PubMed1999

Heat capacity effects on the melting of DNA. 2. Analysis of nearest-neighbor base pair effects.

The stability of a DNA double helix of any particular sequence is conventionally estimated as the average of the stabilities of the 10 different nearest-neighbor (NN) base pair doublets that it contains. Therefore, much effort has been devoted to the experimental characterization and tabulation of the enthalpy, entropy, and free energy of melting for each of the NN doublets. Although 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 the absence of longer range interactions, the length dependence of DNA melting parameters per base pair, the applicability of polyelectrolyte theory to the description of salt effects on oligomers, and the 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.

Base Pairing↗
PubMed1999

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.

Kinetics↗
PubMed1999

Osteoarthritis-susceptibility locus on chromosome 11q, detected by linkage.

We present a two-stage genomewide scan for osteoarthritis-susceptibility loci, using 481 families that each contain at least one affected sibling pair. The first stage, with 272 microsatellite markers and 297 families, involved a sparse map covering 23 chromosomes at intervals of approximately 15 cM. Sixteen markers that showed evidence of linkage at nominal P</=.05 were then taken through to the second stage, with an additional 184 families. This second stage confirmed evidence of linkage for markers on chromosome 11q. Additional markers from this region were then typed to create a denser map. We obtained a maximum single-point LOD score, at D11S901, of 2.40 (P=.0004) and a maximum multipoint-LOD score of 3.15, between markers D11S1358 and D11S35. A subset of 196 of the 481 families, comprising affected female sibling pairs, generated a corrected LOD score of 2.54 (P=.0003) for marker D11S901, with evidence for linkage extending 12 cM proximal to this marker. When we stratified for affected male sibling pairs there was no evidence of linkage to chromosome 11. Our data suggest that a female-specific susceptibility gene for idiopathic osteoarthritis is located on chromosome 11q.

Arthroplasty, Replacement↗
PubMed1998

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.

ADP-ribosyl Cyclase↗
PubMed1998

Insertion of telomere repeat sequence decreases plasmid DNA condensation by cobalt (III) hexaammine.

Telomere repeat sequence (TRS) DNA is found at the termini of most eukaryotic chromosomes. The sequences are highly repetitive and G-rich (e.g., [C(1-3)A/TG(1-3)]n for the yeast Saccharomyces cerevisiae) and are packaged into nonnucleosomal protein-DNA structures in vivo. We have used total intensity light scattering and electron microscopy to monitor the effects of yeast TRS inserts on in vitro DNA condensation by cobalt (III) hexaammine. Insertion of 72 bp of TRS into a 3.3-kb plasmid depresses condensation as seen by light scattering and results in a 22% decrease in condensate thickness as measured by electron microscopy. Analysis of toroidal condensate dimensions suggests that the growth stages of condensation are inhibited by the presence of a TRS insert. The depression in total light scattering intensity is greater when the plasmid is linearized with the TRS at an end (39-49%) than when linearized with the TRS in the interior (18-22%). Circular dichroism of a 95-bp fragment containing the TRS insert gives a spectrum that is intermediate between the A-form and B-form, and the anomalous condensation behavior of the TRS suggests a noncanonical DNA structure. We speculate that under conditions in which the plasmid DNA condenses, the telomeric insert assumes a helical geometry that is similar to the A-form and is incompatible with packing into the otherwise B-form lattice of the condensate interior.

Base Sequence↗
PubMed1998

DNA bending by small, mobile multivalent cations.

We propose a purely electrostatic mechanism by which small, mobile, multivalent cations can induce DNA bending. A multivalent cation binds at the entrance to the B-DNA major groove, between the two phosphate strands, electrostatically repelling sodium counterions from the neighboring phosphates. The unscreened phosphates on both strands are strongly attracted to the groove-bound cation. This leads to groove closure, accompanied by DNA bending toward the cationic ligand. We explicitly treat the dynamic character of the cation-DNA interaction using an adiabatic approximation, noting that DNA bending is much slower than the diffusion of nonspecifically bound, mobile cations. We make semiquantitative estimates of the free energy components of bending-electrostatic (with a sigmoidal distance-dependent dielectric function), elastic, and entropic cation localization-and find that the equilibrium state is bent B-DNA stabilized with a self-localized cation. This is a bending polaron, formation of which should be critically dependent on the strength of electrostatic interaction and the concentration of highly mobile cations available for self-localization. We predict that the resultant bend will be large (approximately 20-40 degrees), smooth (because it is spread over 6 bp), and infrequent. The stability of such a bend can be variable, from transient to highly stable (static) bending, observable with standard curvature-measuring techniques. We further predict that this bending mechanism will have an unusual sequence dependence: sequences with less binding specificity will be more bent, unless the specific binding site is in the major groove.

Base Sequence↗
PubMed1997

Ionic effects on the elasticity of single DNA molecules.

We used a force-measuring laser tweezers apparatus to determine the elastic properties of lambda-bacteriophage DNA as a function of ionic strength and in the presence of multivalent cations. The electrostatic contribution to the persistence length P varied as the inverse of the ionic strength in monovalent salt, as predicted by the standard worm-like polyelectrolyte model. However, ionic strength is not always the dominant variable in determining the elastic properties of DNA. Monovalent and multivalent ions have quite different effects even when present at the same ionic strength. Multivalent ions lead to P values as low as 250-300 A, well below the high-salt "fully neutralized" value of 450-500 A characteristic of DNA in monovalent salt. The ions Mg2+ and Co(NH3)63+, in which the charge is centrally concentrated, yield lower P values than the polyamines putrescine2+ and spermidine3+, in which the charge is linearly distributed. The elastic stretch modulus, S, and P display opposite trends with ionic strength, in contradiction to predictions of macroscopic elasticity theory. DNA is well described as a worm-like chain at concentrations of trivalent cations capable of inducing condensation, if condensation is prevented by keeping the molecule stretched. A retractile force appears in the presence of multivalent cations at molecular extensions that allow intramolecular contacts, suggesting condensation in stretched DNA occurs by a "thermal ratchet" mechanism.

Cobalt↗
PubMed1997

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.

Journal Article↗
PubMed1997

DNA condensation by multivalent cations.

In the presence of multivalent cations, high molecular weight DNA undergoes a dramatic condensation to a compact, usually highly ordered toroidal structure. This review begins with an overview of DNA condensation: condensing agents, morphology, kinetics, and reversibility, and the minimum size required to form orderly condensates. It then summarizes the statistical mechanics of the collapse of stiff polymers, which shows why DNA condensation is abrupt and why toroids are favored structures. Various ways to estimate or measure intermolecular forces in DNA condensation are discussed, all of them agreeing that the free energy change per base pair is very small, on the order of 1% of thermal energy. Experimental evidence is surveyed showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions. The various intermolecular forces whose interplay gives rise to DNA condensation are then reviewed. The entropy loss upon collapse of the expanded wormlike coil costs free energy, and stiffness sets limits on tight curvature. However, the dominant contributions seem to come from ions and water. Electrostatic repulsions must be overcome by high salt concentrations or by the correlated fluctuations of territorially bound multivalent cations. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Undulations of the DNA in its confined surroundings extend the range of the electrostatic forces. The condensing ions may also subtly modify the local structure of the double helix.

Cations↗
PubMed1996

DNA melting investigated by differential scanning calorimetry and Raman spectroscopy.

Thermal denaturation of the B form of double-stranded DNA has been probed by differential scanning calorimetry (DSC) and Raman spectroscopy of 160 base pair (bp) fragments of calf thymus DNA. The DSC results indicate a median melting temperature Tm = 75.5 degrees C with calorimetric enthalpy change delta Hcal = 6.7 kcal/mol (bp), van't Hoff enthalpy change delta HVH = 50.4 kcal/mol (cooperative unit), and calorimetric entropy change delta Scal = 19.3 cal/deg.mol (bp), at the experimental conditions of 55 mg DNA/ml in 5 mM sodium cacodylate at pH 6.4. The average cooperative melting unit (nmelt) comprises 7.5 bp. The Raman signature of 160 bp DNA is highly sensitive to temperature. Analyses of several conformation-sensitive Raman bands indicate the following ranges for thermodynamic parameters of melting: 43 < delta HVH < 61 kcal/mol (cooperative unit), 75 < Tm < 80 degrees C and 6 < (nmelt) < 9 bp, consistent with the DSC results. The changes observed in specific Raman band frequencies and intensities as a function of temperature reveal that thermal denaturation is accompanied by disruption of Watson-Crick base pairs, unstacking of the bases and disordering of the B form backbone. These three types of structural change are highly correlated throughout the investigated temperature range of 20 to 93 degrees C. Raman bands diagnostic of purine and pyrimidine unstacking, conformational rearrangements in the deoxyribose-phosphate moieties, and changes in environment of phosphate groups have been identified. Among these, bands at 834 cm-1 (due to a localized vibration of the phosphodiester group), 1240 cm-1 (thymine ring) and 1668 cm-1 (carbonyl groups of dT, dG and dC), are shown by comparison with DSC results to be the most reliable quantitative indicators of DNA melting. Conversely, the intensities of Raman marker bands at 786 cm-1 (cytosine ring), 1014 cm-1 (deoxyribose ring) and 1092 cm-1 (phosphate group) are largely invariant to melting and are proposed as appropriate standards for intensity normalizations.

Animals↗
PubMed1996

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.

Biophysical Phenomena↗