Simple model for DNA adsorption onto a mica surface in 1:1 and 2:1 electrolyte solutions.

We propose a simple theory of interactions between like-charged polyelectrolyte and a surface based on a mean-field Derjaguin-Landau-Verwey-Overbeek approach. It predicts that the van der Waals attractive interactions are responsible for irreversible physisorption of polyelectrolytes onto charged surfaces. We show that monovalent salts contribute significantly to repulsive interactions, while enhancing the attraction very slightly. The effect of the divalent counterions is reverse. Therefore, to achieve the adsorption, the overall repulsion due to 1:1 electrolyte should be counterbalanced by the stronger van der Waals attraction due to the presence of doubly charged counterions in solution. The theory has been validated experimentally against its ability to predict the minimum polymer/surface interaction energy required for the adsorption using DNA/mica in NaCl, MgCl2, and NiCl2 solutions as a test system. The theory explains the mechanism of linear DNA adsorption to a mica surface for different solvent compositions and can be used as a tool for predicting the optimum conditions for AFM experiments on linear polymer systems. The model can also be used to make general conclusions on the conformation of polymer molecules on a surface. We have shown for the DNA/mica surface system that when the adsorption of DNA is mostly governed by long-range van der Waals forces the molecule adopts an ideal 2D conformation. When the adsorption is mostly due to short-range ion-correlation forces, DNA will appear 3D --> 2D projected in agreement with experimental data.

High and low oxygen affinity conformations of T state hemoglobin.

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15 degrees C, the oxygen pressure at half saturation (p50) was measured to be 12.4 +/- 0.2 and 139 +/- 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 +/- 0.02 and 0.93 +/- 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen-affinity tertiary T-state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of alpha- and beta-hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen-affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high-affinity T state and almost absent for the low-affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10-fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T-state cooperativity observed in solution and in the crystalline state might arise from a ligand-induced perturbation of the distribution between the high- and low-affinity T-state conformations.

Accurate length determination of DNA molecules visualized by atomic force microscopy: evidence for a partial B- to A-form transition on mica.

Achieving the most correct estimate of the contour length of digitized DNA molecules is a key aspect of the microscopic analysis of nucleic acids by either electron microscopy (EM) or atomic force microscopy (AFM). Six different methods, that are mathematically not too complex and suited for common, practical use, have been tested here using simulated polymers in two dimensions and real DNA molecules (564, 1054, 2049 and 4297 bp long) imaged in air by AFM. The main result is that the frequently used Freeman estimator (L(F) = n(e) + square root 2n(o)) overestimates the real contour length of the polymers by about 4%. More accurate estimates are obtained with the Kulpa estimator (L(K) = 0.948n(e) + 1.343n(o)) or with the corner count estimator (L(C) = 0.980)n(e) + 1.406n(o) - 0.091n(c)). In the range of the DNA sizes and magnifications we have considered, however, the best results are obtained with an ad hoc developed routine that smoothes the DNA trace by a polynomial fitting of degree 3 over a moving window of 5 points. Under these conditions, the difference between the measured and the real contour length of the molecules is less than 0.4%. The accuracy of this procedure allowed us to reveal a discrete, size-dependent, shortening of DNA molecules deposited onto mica under low salt conditions and imaged in air by AFM. Awareness of this structural alteration, that can be attributed to a partial transition from B- to A-form DNA, may lead to a more correct interpretation of DNA molecules or protein-DNA complexes imaged by AFM.

Differential expression of transforming growth factors-beta1, -beta2 and -beta3 in human colon carcinoma.

Transforming growth factor (TGF)-beta is a protein family which affects multiple cellular functions including survival, proliferation, differentiation and adhesion. Among the three known isoforms, TGF-beta1 is commonly overexpressed in solid malignancies. Recent studies in knock-out mice demonstrated non-redundant roles of different TGF-beta isoforms in development. The present study was performed to assess tumour-associated expression of the three TGF-beta isoforms in colon carcinoma. We report that colon carcinoma progression is associated with gradual and significant increases in expression of TGF-beta1 and TGF-beta2 mRNA and proteins. By contrast, TGF-beta3 expression was detected in normal colonic mucosa and, at slightly higher levels, in tumour tissues. In addition, plasma levels of both TGF-beta1 and TGF-beta2 were significantly higher in cancer patients when compared with unaffected individuals. Taken together, our results indicate distinct expression patterns of the three TGF-beta isoforms in colon carcinoma cells and possible systemic effects of TGF-beta1 and TGF-beta2 in tumour patients.

Direct observation of one-dimensional diffusion and transcription by Escherichia coli RNA polymerase.

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Wrapping of DNA around the E.coli RNA polymerase open promoter complex.

High-resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase.sigma(70) (RNAP) open promoter complex (RP(o)). A detailed analysis of a large number of molecules shows that the DNA contour length of RP(o) is reduced by approximately 30 nm (approximately 90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88 degrees. The contour length reduction and the DNA bend angle were much less in inactive RNAP-DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300 degrees around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two-thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one-third is part of the downstream region. Based on these data, a model of the sigma(70).RP(o) conformation is proposed.

Polymer chain statistics and conformational analysis of DNA molecules with bends or sections of different flexibility.

The worm-like chain model has often been employed to describe the average conformation of long, intrinsically straight polymer molecules, including DNA. The present study extends the applicability of the worm-like chain model to polymers containing bends or sections of different flexibility. Several cases have been explicitly considered: (i) polymers with a single bend; (ii) polymers with multiple coplanar bends; (iii) polymers with two non-coplanar bends; and (iv) polymers comprised of sections with different persistence lengths. Expressions describing the average conformation of such polymers in terms of the mean-square end-to-end distance have been derived for each case. For cases (i) and (iv), expressions for the projection of the end-to-end vector onto the initial orientation of the chain are presented. The expressions derived here have been used to investigate DNA molecules with sequence-induced bending (A-tracts). Mean-square end-to-end distance values determined from a large number of A-tract containing DNA molecules visualized by scanning force microscopy resulted in an average bend angle of 13.5 degrees per A-tract. A similar study was performed to characterize the flexibility of double-strandedDNA molecules containing a single-stranded region. Analysis of their mean-square end-to-end distance yielded a persistence length of 1.3 nm for single-stranded DNA.

X-ray and spectrophotometric studies of the binding of proflavin to the S1 specificity pocket of human alpha-thrombin.

Proflavin can be used to study the interactions of inhibitors and substrates with thrombin by monitoring the changes in the visible absorption spectrum that occur on dye displacement. We have used microspectrophotometric methods to investigate the binding of proflavin to crystals of an alpha-thrombin-hirugen complex and have determined the structure by X-ray crystallography. The proflavin molecule binds in the S1 pocket of the enzyme with one of the amino groups hydrogen bonded to the carboxylate of Asp-189 while the protonated ring nitrogen is hydrogen bonded to the carbonyl of Gly-219. This result indicates that the proflavin displacement assay can be used to specifically monitor the binding of inhibitors to the S1 pocket.

Scanning force microscopy under aqueous solutions.

Merely ten years after its invention, the scanning force microscope is becoming a powerful method to investigate the structure and dynamics of biological molecules under aqueous environments. From the visualization of transcription in real time to the mechanical manipulation of individual proteins, the advances made during the past year open up a vast number of exciting applications of this technique in biology.

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).

Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis.

This paper reports a study of the deposition process of DNA molecules onto a mica surface for imaging under the scanning force microscope (SFM). Kinetic experiments indicate that the transport of DNA molecules from the solution drop onto the surface is governed solely by diffusion, and that the molecules are irreversibly adsorbed onto the substrate. A statistical polymer chain analysis has been applied to DNA molecules to determine the deposition conditions that lead to equilibrium and those that result in trapped configurations. Using the appropriate conditions, DNA molecules deposited onto freshly cleaved mica, are able to equilibrate on the surface as in an ideal two-dimensional solution. A persistence length of 53 nm was determined from those molecules. DNA fragments that were labeled on both ends with a horseradish peroxidase streptavidin fusion protein were still able to equilibrate on the surface, despite the additional protein-surface interaction. In contrast, DNA molecules deposited onto glow-discharged mica or H+-exchanged mica do not equilibrate on the surface. These molecules adopt conformations similar to those expected for a simple projection onto the surface plane, suggesting a process of kinetic trapping. These results validate recent SFM application to quantitatively analyze the conformation of complex macromolecular assemblies deposited on mica. Under equilibration conditions, the present study indicates that the SFM can be used to determine the persistence length of DNA molecules to a high degree of precision.

Cooperative oxygen binding to scapharca inaequivalvis hemoglobin in the crystal.

Oxygen binding to homodimeric Scapharca inaequivalvis hemoglobin (HbI) crystals has been investigated by single-crystal polarized absorption microspectrophotometry. The saturation curve, characterized by a Hill coefficient nH = 1.45 and an oxygen pressure at half saturation p50 = 4.8 torr, at 15 degrees C, shows that HbI in the crystalline state retains positive cooperativity in ligand binding. This finding will permit the correlation of the oxygen-linked conformational changes in the crystal with the expression of cooperativity. Polarized absorption spectra of deoxy-HbI, oxy-HbI, and oxidized HbI crystals indicate that oxygenation does not induce heme reorientation, whereas oxidation does. Lattice interactions prevent the dissociation of oxidized dimers that occurs in solution and stabilize an equilibrium distribution of pentacoordinate and hexacoordinate high spin species.

Structure and oxygen affinity of crystalline desArg141 alpha human hemoglobin A in the T state.

The correlation of a protein structure determined crystallographically to its functional properties determined in solution can be an extremely complex problem due to potential differences of protein conformational flexibility in the two physical states. A more direct approach to the correlation of structure with function is to examine both the structure and the function of a protein in the same crystalline environment. In this paper, the structural and functional properties of T state desArg hemoglobin (human hemoglobin modified by removal of the alpha-chain COOH-terminal residue, Arg141 alpha) have been studied in the same crystal form by high resolution X-ray diffraction methods and by polarized absorption microspectrophotometry. Specifically, the crystal structure of deoxygenated desArg human hemoglobin has been refined at a 2.1 A resolution using crystals grown at low salt concentration from solutions of polyethylene glycol. The loss of Arg141 alpha and all of the salt bridges in which it participates is associated with subtle structural perturbations of the alpha-chains which include an increase in the conformational flexibility of both the NH2 and COOH-terminal peptides. Although the heme pockets appear unchanged and even the side-chain of Tyr140 is oriented nearly as in HbA, the functional characterization by microspectrophotometric measurements indicates that crystals of desArg hemoglobin bind oxygen with an affinity which is roughly 15-fold greater than that of crystals of human hemoglobin A. There is no alkaline Bohr effect or effect of chloride ions, but an acid Bohr effect is observed. The oxygen affinities measured along two principal axes of the crystals differ by 25%, indicating heterogeneity in the affinities of the oxygen binding sites. This finding and the measured Hill coefficient of unity suggest significant cooperativity in the binding of oxygen in these crystals. The origins of the observed heterogeneity and the implied cooperativity are unknown.

Effect of chloride on oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure.

Oxygen binding to crystals of hemoglobin Rothschild (beta 37 Trp-->Arg) in the T quaternary structure has been investigated by polarized absorption microspectrophotometry. These crystals were grown from poly(ethylene glycol) solutions containing low concentrations of salt. In the absence of chloride, they have a significantly higher oxygen affinity than crystals of human hemoglobin A grown in a similar manner, and exhibit Hill coefficients lower than 1. There is no Bohr effect from pH 6 to 9. We have found that chloride decreases the oxygen affinity of Hb Rothschild crystals, an effect which is absent in crystals of HbA. This dependence of affinity on chloride is almost certainly associated with the chloride binding sites which have been localized crystallographically at the mutant arginine residues (Kavanaugh et al., 1992). Since chloride binding appears to lower the oxygen affinities of both the alpha and beta chains, the linkage between the binding of oxygen and the dissociation of chloride results in significant cooperativity in oxygen binding to the crystals.

Oxygen binding by single crystals of hemoglobin.

Reversible oxygen binding curves for single crystals of hemoglobin in the T quaternary structure have been measured using microspectrophotometry. Saturations were determined from complete visible spectra measured with light linearly polarized parallel to the a and c crystal axes. Striking differences were observed between the binding properties of hemoglobin in the crystal and those of hemoglobin in solution. Oxygen binding to the crystal is effectively noncooperative, the Bohr effect is absent, and there is no effect of chloride ion. Also, the oxygen affinity is lower than that of the T quaternary structure in solution. The absence of the Bohr effect supports Perutz's hypothesis on the key role of the salt bridges, which are known from X-ray crystallography to remain intact upon oxygenation. The low affinity and absence of the Bohr effect can be explained by a generalization of the MWC-PSK model (Monod, Wyman, & Changeux, 1965; Perutz, 1970; Szabo & Karplus, 1972) in which both high- and low-affinity tertiary conformations, with broken and unbroken salt bridges, respectively, are populated in the T quaternary structure. Because the alpha and beta hemes make different projections onto the two crystal axes, separate binding curves for the alpha and beta subunits could be calculated from the two measured binding curves. The approximately 5-fold difference between the oxygen affinities of the alpha and beta subunits is much smaller than that predicted from the crystallographic study of Dodson, Liddington, and co-workers, which suggested that oxygen binds only to the alpha hemes.(ABSTRACT TRUNCATED AT 250 WORDS)

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c-type cytochrome.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.

Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect.

The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.