Quaternary and quinternary structures of native chromatin DNA in liver nuclei: differential scanning calorimetry.

Differential scanning calorimetry of chromatin isolated from rat liver cells revealed three discrete thermal transitions whose temperatures and melting enthalpies depend on ionic strength in the range 0 to 600 millimolar NaCl. Intact nuclei showed a fourth thermal transition at a lower temperature and different melting enthalpies for the other three transitions still present at temperatures similar to those obtained in isolated chromatin. The data are discussed in terms of the tertiary, quaternary, and quinternary structures of chromatin DNA.

Solution structure of human plasma fibronectin under different solvent conditions. Fluorescence energy transfer, circular dichroism and light-scattering studies.

Human plasma fibronectin is a high molecular weight (530,000), multi-domain, modular glycoprotein, consisting of two nearly identical subunits disulfide-bridged close to their C-terminal ends. Three sites that can be differentially labeled with fluorescent probes are present on each fibronectin subunit, the transglutaminase-sensitive Gln3 residue and the two free sulfhydryl residues, Cys1201 and Cys2196. These sites are located, respectively, in the N-terminal heparin/fibrin-binding domain, between the central DNA and cell-binding domains, and just before the C-terminal fibrin-binding domain. To map the relative spatial arrangement of these domains, steady-state and lifetime fluorescence energy transfer techniques were employed. Our results show that the minimal intramolecular distances between the labeled Gln3-Cys1201 and Gln3-Cys2196 pairs are 5.5(+/- 0.6) nm and 5.7(+/- 0.7) nm, respectively, as measured by steady-state methods. Lifetime methods gave somewhat higher distances of 8.1(+/- 0.2) nm and 7.6(+/- 0.2) nm, respectively, between these sites. The binding of heparin or subjection to high ionic strength had only a minor effect, while in the presence of 50% (w/v) glycerol, an increase of about 25% in the intramolecular distances between these sites was observed. A similar effect was induced by binding of fibronectin to the surface of Cytodex beads, an event which was previously shown instead to markedly increase the intersubunit distances between the Gln3-Gln3 and Cys1201-Cys1201 pairs. The solution structure of fibronectin was further investigated by elastic light-scattering and circular dichroism measurements. By elastic light-scattering, the radius of gyration of fibronectin was found to be 15.3(+/- 0.8) nm in the presence of 30% (w/v) glycerol, in contrast to a value of 8.6(+/- 0.3) nm under physiological conditions. Far and near ultraviolet circular dichroism spectra showed that only minor changes in the secondary structure of fibronectin take place on increasing the glycerol content of the solvent up to 34% (w/v). Our results complement previously available information on the solution structure of fibronectin and on its transition from the native compact conformation to a more expanded form on increasing ionic strength or glycerol content. In either situation, fibronectin seems to retain a basic structural core, in which the N-terminal, the central and the C-terminal regions of the two subunits strongly interact with each other. A major role of hydrophobic forces, in stabilizing the fibronectin conformations under these conditions, is therefore postulated. The transition to the extended forms seen in many electron micrographs can instead be explained by disruption of the proposed structural core upon adsorption to surfaces.

Mode-coupling smoluchowski dynamics of a double-stranded DNA oligomer

The local dynamics of a double-stranded DNA d(TpCpGpCpG)(2) is obtained to second order in the mode-coupling expansion of the Smoluchowski diffusion theory. The time correlation functions of bond variables are derived and the (13)C-nmr spin-lattice relaxation times T(1) of different (13)C along the chains are calculated and compared to experimental data from the literature at three frequencies. The DNA is considered as a fluctuating three-dimensional structure undergoing rotational diffusion. The fluctuations are evaluated using molecular dynamics simulations, with the ensemble averages approximated by time averages along a trajectory of length 1 ns. Any technique for sampling the configurational space can be used as an alternative. For a fluctuating three-dimensional (3D) structure using the three first-order vector modes of lower rates, higher order basis sets of second-rank tensor are built to give the required mode coupling dynamics. Second- and even first-order theories are found to be in close agreement with the experimental results, especially at high frequency, where the differences in T(1) for (13)C in the base pairs, sugar, and backbone are well described. These atomistic calculations are of general application for studying, on a molecular basis, the local dynamics of fluctuating 3D structures such as double-helix DNA fragments, proteins, and protein-DNA complexes. Copyright 1999 John Wiley & Sons, Inc.

Early events in the polymerization of fibrin.

The early events in the thrombin-induced formation of fibrin have been studied by the use of stopped-flow multiangle laser light scattering (SF-MALLS). This technological advancement has allowed the recovering, as a function of time with a resolution of about 0.5 sec, of the mean square radius of gyration (Rg2)z and of the molecular weight Mw, and to place an upper bound to the values of the mass/unit length ML. The ionic strength, pH and salt type conditions investigated were all close to physiological, starting with a 50 mM Tris, 104 mM NaCl, pH 7.4 buffer (TBS), to which either 1 mM EDTA-Na2 or 2.5 mM CaCl2 were also added. Fibrinogen was 0.2-0.3 mg/ml and rate-limiting concentrations of thrombin were used (0.05-0.25 NIH units/mg fibrinogen). By plotting (Rg2)z and ML versus Mw on log-log scales, runs proceeding at different velocities and under different solvent conditions could be compared and confronted with model curves. It was found that: (1) within this thrombin range, the mechanism of association does not depend on its concentration, nor on the buffers employed; (2) the (Rg2)z versus Mw curves could all be reasonably fitted with a bifunctional polycondensation scheme involving semiflexible worm-like, double-stranded, half-staggered polymers with persistence length between 200-600 nm, provided that a ratio Q = 16 between the rate of release of the two fibrinopeptides A was employed; (3) the ML versus Mw data seemed more compatible with lower Q values (4 < Q < 8), but their uncertainty prevented a better assessment of this issue; the formation of fibrinogen-fibrin monomer complexes may also play a role in the polymer distributions; (4) in the very early stages (e.g., when Mw < 7 x 10(5)), the (Rg2)z versus Mw data were fitted well only in TBS and at the lowest thrombin concentration, suggesting that a transient, either sequential or concurrent fast second mechanism, involving longer and thinner polymers, may be at work.

Environment-induced changes in DNA conformation as probed by ethidium bromide fluorescence.

The interaction of the ethidium cation with calf thymus DNA is investigated in solutions of different ionic strength and temperature by observation of the enhancement of fluorescence of ethidium upon intercalation in the duplex structure. The quantum yield of the fluorescence of the intercalated dye is found to increase either upon lowering the Na+ concentration or upon increasing the temperature. The existence of a correlation between the geometry of the intercalation complex and the features of the secondary structure of DNA is suggested. Binding isotherms under corresponding environmental conditions are also quantitated by fluorescence enhancement and interpreted in terms of the neighbor exclusion model. Large contributions from change in hydration to the thermodynamics of binding are demonstrated by the temperature dependences of the equilibrium constants. The neighbor exclusion range is found to be practically independent of the salt concentration but its value increases from an average of 2.4 around room temperature to 4-5 at 80 degrees C, as inferred from the binding curves in 0.15 and 0.5 M [Na+] or from the DNA hypochromism vs temperature profiles of complexes at 10(-3) M [Na+]. All the data point to a possible sequence-conformation specificity in the intercalation of ethidium which in heterogeneous DNA is mediated by environmental changes.

Self-assembly of fibrin monomer. A light scattering and electron microscopic investigation.

A light scattering method, together with complementary electron microscopy observations, was designed to investigate the self-assembly of fibrin. Calcium-free monomer was used, and clot reconstitution was carried out in solvents corresponding to limit interaction energies of the protein with the medium. The self-assembly process, under physiological conditions, conforms to the following sequence of events. 1) A fast polymerization step leading to linear aggregates. 2) Fiber growth; at this stage onset of the network occurs. 3) Gelation (clot formation). Bound calcium was found to be structurally required for gelation. Its removal results in the formation of thick fibers, which are unable to clot. Evidence is reported favouring our previous hypothesis (Conio et al., 1976) on the onset of the network: branching of linear aggregates is a prerequisite for clotting. The occurrence of a crystallization process, which overlaps to fiber growth, is demonstrated in this paper for the first time. Its dependence on solvent-protein interactions is analyzed. Our results suggest that fibrin monomer is to some extent a flexible molecule. Both flexibility and crystallization may play a functional role in the clotting process in vivo.

Models of fibronectin.

The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/- 0.8 nm and 10.7 +/- 0.9 nm respectively, for a constant mol. wt of 533,000 +/- 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end-to-end joined chains of 28 beads, was then constructed, and their calculated physico-chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino-terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy-terminal fibrin(ogen)-binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.

Polymerization of rod-like macromolecular monomers studied by stopped-flow, multiangle light scattering: set-up, data processing, and application to fibrin formation.

Many biological supramolecular structures are formed by polymerization of macromolecular monomers. Light scattering techniques can provide structural information from such systems, if suitable procedures are used to collect the data and then to extract the relevant parameters. We present an experimental set-up in which a commercial multiangle laser light scattering photometer is linked to a stopped-flow mixer, allowing, in principle, the time-resolved extrapolation of the weight-average molecular weight M(w) and of the z-average square radius of gyration (z) of the polymers from Zimm-like plots. However, if elongated structures are formed as the polymerization proceeds, curved plots rapidly arise, from which M(w) and (z) cannot be recovered by linear fitting. To verify the correctness of a polynomial fitting procedure, polydisperse collections of rod-like or worm-like particles of different lengths, generated at various stages during bifunctional polycondensations of rod-like macromolecular monomers, were considered. Then, the angular dependence of their time-averaged scattered intensity was calculated in the Rayleigh-Gans-Debye approximation, with random and systematic noise also added to the data. For relatively narrow size distributions, a third-degree polynomial fitting gave satisfactory results across a broad range of conversion degrees, yielding M(w) and (z) values within 2% and no greater than 10-20%, respectively, of the calculated values. When more broad size distributions were analyzed, the procedure still performed well for semiflexible polymers, but started to seriously underestimate both M(w) and (z) when rigid rod-like particles were analyzed, even at relatively low conversion degrees. The data were also analyzed in the framework of the Casassa approximation, from which the mass per unit length of the polymers can be derived. These procedures were applied to a set of data taken on the early stages of the thrombin-catalyzed polymerization of fibrinogen, a rod-like macromolecule approximately 50 nm long. The polymers, grown in the absence of Ca(2+) by rate-limiting amounts of thrombin, appeared to be characterized by a much broader size distribution than the one expected for a classical Flory bifunctional polycondensation, and they seem to behave as relatively flexible worm-like double-stranded chains. Evidence for the formation of fibrinogen-fibrin monomer complexes is also inferred from the time dependence of the mass/length ratio. However, our data are also compatible with the presence of limited amounts of single-stranded structures in the very early stages, either as a secondary, less populated pathway, or as transient intermediates to the classical double-stranded fibrils.

Renal selectivity properties towards endogenous albumin in minimal change nephropathy.

It is well accepted that the molecular charge and conformation of serum proteins are major determinants of their glomerular filtration, but few studies characterizing the molecular features of circulating proteins in renal diseases are currently available. In 11 children affected by minimal change nephropathy (MCN) we determined the electrical charge and the fluorescence quantum yield of Tyrosine (Tyr) and Tryptophan (Trp) (taken as index of conformation) of serum and urinary albumin before and after steroid-induced remission of proteinuria. In all proteinuric children at the onset of the disease, urinary albumin was formed by one band with an isoelectric point (pI) of 4.7 (pI of the native protein), and by numerous other, less anionic bands with pIs between 4.8 and 5.5 accounting for about 50% of the total amount of this protein. The normalization of proteinuria which followed steroid therapy was characterized by the disappearance in urines of the less anionic fraction and by the appearance of numerous isoforms with a pI still more anionic (pI less than 4.7) than normal. At the same time, in the proteinuric phase, the fluorescence quantum yield of Trp of urinary albumin was markedly quenched, returning to near normal levels after steroid-induced remission of proteinuria. These data indicate that in MCN the charge-dependent renal selectivity properties are partially maintained and that the less anionic isoforms of albumin are a main component of urinary albumin. Together with the electrical charge, the conformation of albumin as a major determinant of its urinary excretion in MCN must also be considered.

Backbone dynamics for the wild type and a double H52R/T56W mutant of the vnd/NK-2 homeodomain from Drosophila melanogaster.

The (15)N relaxation behavior and heteronuclear Overhauser effect data for the wild type and an H52R/T56W double mutant protein that encompasses the vnd/NK-2 homeodomain from Drosophila melanogaster were used to characterize and describe the protein backbone dynamics. This investigation, which includes a description of a model structure for the H52R/T56W double mutant vnd/NK-2 homeodomain, was carried out for the two proteins in both the free and DNA-bound states. The double residue replacement at positions 52 and 56 within the DNA recognition helix of vnd/NK-2 has been shown to lead to a significant secondary structural modification resulting in an increase in the length of the recognition helix for the unbound protein. These structural changes are accompanied by corresponding changes in the T(1) and T(1)(rho) relaxation times as well as in the heteronuclear Overhauser effect (XNOE) values that show that the structural stability of the protein is enhanced by the two residue replacements. The values of the rotational anisotropy, D(parallel)/D(perpendicular), derived from analysis of the (15)N T(1) and T(1)(rho) relaxation values are small (1.189 for the unbound homeodomain and 1.110 for the bound homeodomain; both analyzed as prolate ellipsoids of revolution). A comparison of the T(2) values of the wild type and double mutant homeodomain reveals the presence of a low-frequency exchange contribution for the wild type analogue. These relaxation studies show that the motional behavior of the protein primarily reflects the tertiary structure and stability of the homeodomain backbone as well as the respective changes induced upon site-directed residue replacement or DNA binding.