The fibrinogen molecule: its size,shape, and mode of polymerization

Improved electron micrographs of the shadow-cast bovine fibrinogen molecule have been obtained establishing its general morphology and dimensions in the dry state. It consists of a linear array of 3 nodules held together by a very thin thread which is estimated to have a diameter of from 8 to 15 A, though it is not clearly resolved. The two end nodules are alike but the center one is slightly smaller. Measurements of shadow lengths indicate that nodule diameters are in the range 50 to 70 A. The length of the dried molecule is 475 +/- 25 A. Adopting the molecular volume from previous physical chemical data and the general morphological features and length from electron microscopy, we calculate the diameters of the end nodules to be 65 A and the center one as 50 A. The model of the molecule so obtained is consistent with the electron microscopical observations and the data from physical chemistry. The intermediate polymers formed when fibrinogen is activated with thrombin were also examined and found to be end-to-end aggregates of altered fibrinogen molecules which shrink in length during the process. Intermediate polymer lengths are from 1000 to 5000 A. The nodular nature of fibrinogen, its shrinkage and end-to-end aggregation on polymerization permits us to deduce an explanation for the system of cross-bands previously observed in stained fibrin fibrils.     

Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.     

Corneal and scleral type I collagens: analyses of physical properties and molecular flexibility

The physical properties of type I collagen were studied by electron microscopy of rotary shadowed collagen molecules and laser light scattering techniques. The physical properties, molecular structure and flexibility of type I collagen molecules from two structurally and functionally different connective tissues, cornea and sclera, were similar when measured in HCl, pH 2.0. The molecular weights were 328 and 298 × 10² for corneal and scleral type I collagen, respectively, while the values of T_M were 33.7°C for both preparations. These values were in agreement with those obtained for other type I collagens. The higher level of glycosylation in corneal versus scleral type I collagen did not significantly modify the physical properties of type I collagen in acid solution or the charge distribution along the molecule as determined from the positively stained SLS banding patterns. Our morphological studies indicated that the collagen molecule, although relatively flexible based on electron microscopy, behaved as a long thin rod in solution. The mean end-to-end distances measured from electron micrographs were 253 and 256 nm for corneal and cler type I collagen, respectively, while the molecular contour lengths were 298 and 305 nm. The translational diffusion coefficients (0.849 and 0.857 × 10^?7cm²s^?1) were consistent with the contour lengths while the reported values in the literature for the rotational diffusion coefficient of type I collagen were consistent with the end-to-end distances. The intermediate value for molecular length obtained from the particle scattering factor (277 nm) reflects contributions from all possible molecular configurations.     

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.     

AN ELECTRON MICROSCOPE STUDY OF CANINE CARDIAC MYOSIN AND SOME OF ITS AGGREGATES

The morphology of the canine cardiac myosin molecule has been investigated in the electron microscope with Hall's mica-replica technique. The molecule is an elongated rod (shaft) of nonuniform diameter with a globular expansion (head) on one end. Statistical analysis of the lengths of 1908 molecules showed that the mean length was 1610 ± 250 A; the mean length of the head was 210 ± 20 A; and the diameter of the head and that of the shaft were 35 to 40 and 15 to 20 A, respectively. About one-third of the molecules had single or multiple, fairly sharp, angulations along their shafts. Rarely, some details of the substructure of the molecule have been observed. Large, spindle-shaped aggregates, measuring 0.5 to 1 µ in length and 50 to 100 A in diameter, were produced by dilution of the myosin solutions. These aggregates were readily visualized in the electron microscope by means of Huxley's negative-staining technique. Projections often were visible along the length of the aggregates except at a central zone where they were frequently absent. The aggregates resembled the thick myofilaments of the myocardium and appeared similar to those produced by Huxley from skeletal myosin solutions.     

Structure and mobility of actin filaments as measured by quasielastic light scattering, viscometry, and electron microscopy

Actin filaments of different lengths were prepared by polymerizing actin in the presence of various concentrations of gelsolin, a protein which accelerates actin polymerization by stabilizing nuclei from which filaments grow and which binds to their fast growing ends. The lengths of the actin filaments following polymerization were measured by electron microscopy and showed that the number-average filament length agreed with the predicted length if each gelsolin molecule acted as a seed for the growth of an actin filament. The distribution of lengths was independent of the actin:gelsolin ratio and was similar to that of actin filaments polymerized in the absence of gelsolin (Lw/Ln = 1.8). The mobility of these filaments in solution was studied by quasielastic light scattering and by viscometry. The translational diffusion constant determined by quasielastic light scattering was in agreement with the infinite dilution values calculated from the dimensions and the distribution of lengths determined by electron microscopy for relatively short filament lengths. Under conditions where overlap of the rotational domains of the filaments would be expected to occur, the measured diffusion rates deviated from their predicted dilute solution values and the solution viscosity increased abruptly. The dependence of the diffusion constant and the solution viscosity on the length of the actin filaments can be explained in terms of a theory that describes the restraints on diffusion of independent rigid rods in semi-dilute solution. The results suggest that the rheology of actin filaments can be accounted for by steric restraints. The length of cytoplasmic actin filaments in some cell types is such that these steric constraints are significant and could produce large changes in physical properties with small changes in filament length.     

Structural organization of the fibrinogen molecule

A brief review of the chemical structure of the fibrinogen molecule, its proteolytic fragmentation, physicochemical and functional properties of proteolytic fragments is presented. The previous notions on the structure of the fibrinogen molecule are considered as well as new data obtained by the methods of scanning microcalorimetry, electron microscopy and by other methods which permit suggesting some models for structural organization of the fibrinogen molecule.     

Electron microscopic studies of plasmic degradation products of fibrinogen. Implications for the disulfide structure of fibrinogen.

Fibrinogen, coagulable plasmic derivatives (Fragments X) and Fragments Y, D and E were studied by negative staining electron microscopy. Fragment X obtained from Stage 1 digests and fibrinogen were both globular, while Fragment X of Stage 2 digests appeared as a nodular filament. The Stage 1 and Stage 2 Fragment X preparations had approximately the same molecular weight, but could be differentiated by several subtle differences in polypeptide chain structure. Fragments Y and D were also filamentous, although shorter than Fragment X (Stage 2), and Fragment E appeared as a small, compact or folded filament. These results agree with the concept that fibrinogen consists of a strand of nodules connected by thin strands, folded into a compact, spherical shape. The molecule opens up when stabilizing bonds are disrupted or liberated by plasmin. The data are compatible with a fibrinogen molecule in which the two halves are linked by a single locus of disulfide bonds at the amino terminus and with the asymmetric hypothesis of plasmic degradation to Fragments X, Y, D and E.     

THE FILAMENT LATTICE OF COCKROACH THORACIC MUSCLE

The fine structure of the tergo-coxal muscle of the cockroach, Leucophaea maderae, has been studied with the electron microscope. This muscle differs from some other types of insect flight muscles inasmuch as the ratio of thin to thick filaments is 4 instead of the characteristic 3. The cockroach flight muscle also differs from the cockroach femoral muscle in thin to thick filament ratios and diameters and in lengths of thick filaments. A comparison of these latter three parameters in a number of vertebrate and invertebrate muscles suggests in general that the diameters and lengths of the thick filaments and thin to thick filament ratios are related.     

Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible ''naked'' protein regions.

Subunits of human cervical-mucus glycoproteins obtained by reductive cleavage of whole mucins and high-Mr glycopeptides (T-domains) obtained after their trypsin digestion were studied with electron microscopy after spreading the macromolecules in a monolayer of benzyldimethylalkylammonium chloride. Subunits were observed as linear and apparently flexible particles, with number- and weight-average lengths of 390 nm and 460 nm respectively. T-domains randomly distributed on the grid have number- and weight-average lengths of 90 nm and 103 nm respectively, whereas when aligned (possibly stretched by flow) they were longer, with number-average and weight-average lengths of 150 nm and 170 nm respectively. Subunits complexed with gold appeared as segmented structures, with a distribution of inter-gold distances similar to the length distribution for the relaxed T-domains. The whole mucins had few binding sites for gold, suggesting that reduction exposes hydrophobic protein-rich regions with high affinity for gold. Most T-domains had a binding site at one end, indicating the presence of a residual protruding naked peptide region. We conclude that mucins are assembled from subunits joined end-to-end, and that each subunit consists of alternating oligosaccharide 'clusters' (approx. 100 nm) and naked peptide regions which have (after reduction) a high affinity for colloidal gold.     

Abnormal root and nodule vasculature in R50 (sym16), a pea nodulation mutant which accumulates cytokinins

BACKGROUND AND AIMS: R50 (sym16) is a pea nodulation mutant with fewer and shorter lateral roots (LR), fewer nodules and high levels of cytokinins (CK). Because a link exists between CK imbalance and abnormal vasculature, the vasculature of the primary root (PR) and LR of R50 was studied and it was compared with that of the wild-type 'Sparkle'. Also nodule vasculature was investigated to correlate R50 low nodulation phenotype with CK accumulation. METHODS: PR and first-order LR were hand-sectioned transversely in different locations and at different ages. Vascular poles were counted and root and stele diameters measured. To evaluate LR primordia number, roots were cleared. Nodules obtained from inoculated plants were either fixed and sectioned or cleared; numbers of vascular strands and of tracheary elements in the strands were counted. KEY RESULTS: 'Sparkle' PR is triarch, whereas that of R50 can be triarch, tetrarch or pentarch. Furthermore, as the R50 roots developed, supernumerary vascular strands appeared but, as they aged, the new growth of more roots displayed the triarch pattern. LR vasculature differed from that of PR: whereas 'Sparkle' LR had three or four poles, those of R50 had two or three. No differences in PR or PR stele diameters existed between the two lines. Whereas 'Sparkle' nodules had two vascular strands, most R50 nodules possessed three; however, because R50 nodules were variable in size, their vasculature was highly diverse in terms of strand length. A strong correlation was found between nodule length and number of tracheary elements in strands. CONCLUSIONS: R50 displays an additional number of vascular poles in its PR, a smaller number of vascular poles in its first-order LR and an altered vasculature in its nodules. It appears that these three characteristics are linked to the high levels of CKs that the mutant accumulates over its development.     

Crystals of modified fibrinogen: Size,shape and packing of molecules

We have produced several new macroscopic crystal forms and a variety of microcrystals from modified flbrinogens. Bovine and rabbit flbrinogens crystallize after limited digestion by a bacterial protease or α-chymotrypsin. The fibrinogens making up these crystals are largely intact and highly clottable. Tentative molecular packing arrangements for two crystal forms have been deduced. The crystal morphology and cleavage planes were used in this analysis. The characteristic α-helical coiled-coil reflections arid spikes of intensity in certain directions in the crystal X-ray patterns serve as markers for the orientation of the fibrinogen molecules. Changes that occur in one of the forms during preparation for electron microscopy, as shown by comparison with X-ray experiments on crystals in various stains and solvents, support this packing model. These studies provide preliminary evidence that fibrinogen is about 450 Å in length and that the molecules bond end-to-end to form filaments making up the crystals.     

Structure, force, and energy of a double-stranded DNA oligonucleotide under tensile loads

The end-to-end stretching of a duplex DNA oligonucleotide has been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) simulations and atomic force microscopy (AFM) experiments. Near quantitative agreement between the calculations and experiments was obtained for both the extension length and forces associated with strand separation. The PMF calculations show that the oligonucleotide extends without a significant energetic barrier from a length shorter than A-DNA to a length 2.4 times the contour length of B-DNA at which the barrier to strand separation is encountered. Calculated forces associated with the barrier are 0.09±0.03 nN, based on assumptions concerning tip and thermal-activated barrier crossing contributions to the forces. Direct AFM measurements show the oligonucleotide strands separating at 2.6±0.8 contour lengths with a force of 0.13±0.05 nN. Analysis of the energies from the MD simulations during extension reveals compensation between increases in the DNA-self energy and decreases in the DNA-solvent interaction energy, allowing for the barrierless extension of DNA beyond the canonical B form. The barrier to strand separation occurs when unfavorable DNA interstrand repulsion cannot be compensated for by favorable DNA-solvent interactions. The present combination of single molecule theoretical and experimental approaches produces a comprehensive picture of the free energy surface of biological macromolecular structural transitions. Received: 2 June 1998 / Revised version: 25 January 1999 / Accepted: 11 February 1999     

Electron microscopic measurement of chain flexibility of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II).

The antitumor drug cis-diamminedichloroplatinum (II) (cis-Pt) forms bidentate adducts with guanine residues of poly(dG-dC).poly(dG-dC). The secondary structure of the polymer is altered. In this work, high resolution pictures of naked molecules, obtained by dark field electron microscopy reveal DNA chain distortions with radii as small as 30 A. The extent of distortion increases with the drug/nucleotide ratio (rb). These alterations of the secondary structure are responsible for the apparent shortening of the molecules. Measurements of the persistence lengths of the polymer as well as the end-to-end distances of elementary segments of various lengths, are obtained from digitized electron micrographs. The measurements are used to monitor and quantify the observed modifications of polymer structure upon cis-Pt binding at various rb or incubation times. Poly(dG-m5dC).poly(dG-m5dC) in the B and Z forms have different persistence lengths. In the B form, this polymer is more altered by cis-Pt than in the Z one.     

The separation between the 5′-3′ ends in long RNA molecules is short and nearly constant

RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5′ and 3′ ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5–9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are ‘effectively circularized’ something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation.     

Mechanism of self-assembly of soluble fibrin oligomers and the role of fibrinopeptides A and B in this process

Three-dimensional organization of intermediate soluble forms of fibrin-polymers--a product of fibrin-monomer assembly--in the presence of non-denaturating urea concentrations has been studied. Hydrodynamic parameters of fibrinogen and fibrin-polymers were obtained by viscosimetry, dynamic light scattering and analytic ultracentrifugation. Using Yamakawa's hydrodynamic theory and considering fibrinogen molecule as an oblate ellipsoid of revolution made it possible to estimate the concentration effect on the coefficient of translational friction in the first, according to concentration, linear approximation. Hydrodynamic constants against polymer molecular weights were plotted using Swedberg's and Kuhn--Mark's equation. This made it possible to prove the existence of equilibrium single-stranded protofibrils formed by fibrin-monomer "end-to-end" association. It was concluded that local conformational transformations in fibrin-monomer molecule result in diminishing the complementarity of lateral binding sites; less specific D--D contacts remaining the only means of the "end-to-end" association. Experimentally obtained data give evidence that polymerization region is shifted towards much lower urea concentration, fibrinopeptide B being preserved. Therefore, the possibility of several conformational states of protein molecules during fibrinogen--fibrin transformation is discussed. It is supposed that changes in structure concern not only the central E-, but also the peripheral D- or alpha C-domains.     

Distribution of actin bundles in Bowman's capsule of rat kidney

In this study we define the distribution of actin bundle arrangement in Bowman's capsule of rat renal corpuscles. Parietal cells of Bowman's capsule were examined by conventional light microscopy, electron microscopy and confocal microscopy. Within each parietal cell individual actin bundles are arranged in a parallel fashion running the length of the cell. Computer reconstructions obtained using confocal microscopy clearly show the lengths of actin bundles to be arranged, on a capsule level, end-to-end, at angles and perpendicular to bundles in adjacent cells. The bundles stain positively for non-muscle myosin and vinculin. The presence and arrangement of actin bundles in parietal cells is consistent with a role in reinforcing capsule structure.     

Ultrastructure of the sodium pump: Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membrane-bound (Na+, K+)-ATPase

Purified (Na+, K+)-ATPase was studied by electron microscopy after thin sectioning, negative staining, and freeze-fracturing, particular emphasis being paid to the dimensions and frequencies of substructures in the membranes. Ultrathin sections show exclusively flat or cup-shaped membrane fragments which are triple-layered along much of their length and have diameters of 0.1-0.6 μm. Negative staining revealed a distinct substructure of particles with diameters between 30 and 50 A and with a frequency of 12,500 +/- 2,400 (SD) per μm(2). Comparisons with sizes of the protein components suggest that each surface particle contains as its major component one large catalytic chain with mol wt close to 100,000 and that two surface particles unite to form the unit of (Na+,K+)-ATPase which binds one molecule of ATP or ouabain. The further observations that the surface particles protrude from the membrane surface and are observed on both membrane surfaces in different patterns and degrees of clustering suggest that protein units span the membrane and are capable of lateral mobility. Freeze-fracturing shows intramembranous particles with diameters of 90-110 A and distributed on both concave and convex fracture faces with a frequency of 3,410 +/- 370 per μm(2) and 390 +/- 170 per μm(2), respectively. The larger diameters and three to fourfold smaller frequency of the intramembranous particles as compared to the surface particles seen after negative staining may reflect technical differences between methods, but it is more likely that the intramembranous particle is an oliogomer composed of two or even more of the protein units which form the surface particles.     

Relating Single-Molecule Measurements to Thermodynamics

Measurements made on large ensembles of molecules are routinely interpreted using thermodynamics, but the normal rules of thermodynamics may not apply to measurements made on single molecules. Using a polymer stretching experiment as an example, it is shown that in the limit of a single, short molecule the outcome of experimental measurements may depend on which variables are held fixed and which are allowed to fluctuate. Thus an experiment in which the end-to-end distance of the polymer molecule is fixed and the tension fluctuates yields a different result than an experiment where the force is fixed and the end-to-end distance fluctuates. It is further shown that this difference is due to asymmetry in the distribution of end-to-end distances for a single molecule, and that the difference vanishes in the appropriate thermodynamic limit; that is, as the polymer molecule becomes long compared to its persistence length. Despite these differences, much of the thermodynamic formalism still applies on the single-molecule level if the thermodynamic free energies are replaced with appropriate potentials of mean force. The primary remaining differences are consequences of the fact that unlike the free energies, the potentials of mean force are not in general homogeneous functions of their variables. The basic thermodynamic concepts of an intensive or extensive quantity, and the thermodynamic relationships that follow from them, are therefore less useful for interpreting single-molecule experiments.