Physicochemical studies on xylinan (acetan). I. Characterization by G permeation chromatography on sepharose Cl-2B coupled with static light scattering and viscometry

Laboratory-made samples of the polysaccharide xylinan (acetan) were fractionated on Sepharose Cl-2B using 0.1M NaCl as eluant. The weight average molar masses and intrinsic viscosities were estimated in the fractions by multiangle laser light scattering (off-line) and capillary viscometry, respectively. The Mark-Houwink-Sakurada plot was found to be indicative of semiflexible coils (a = 0.90). The angular dependence of scattered light was interpreted by fitting with theoretically calculated “Master Curves” in terms of a wormlike chain model. The ambiguity of the interpretation of scattering curves owing to the overlapping effects of chain stiffness and polydispersity is discussed in detail. The experimental data is found to be consistent with a persistence length of L_p = 100 nm. The main proportion consists of double-stranded chains (consistent with a robust double-helix), but single- and multistranded chains also are present. Our results suggest a fractionation according to the contour length rather than the molar mass. © 1996 John Wiley & Sons, Inc.     

Electron microscopy of native xanthan and xanthan exposed to low ionic strength

Optical rotation data indicate that xanthan can exist both in an ordered and a disordered conformation. Using molecular weights obtained from light scattering measurements and contour length distributions obtained from electron micrographs, we find that a native, filtered xanthan exposed to low salinity (< 10^?4M NaCl) and subsequently returned to 0.1M NaCl has a highly elongated structure with a mass per unit length of 1950 ± 200 Dalton/nm. Our data thus suggest that the ordered conformation of this xanthan is double stranded. We find that native, filtered xanthan in 0.1M NH₄Ac has a nearly similar structure, but exists in part as aggregates of varying shape and size. Electron micrographs of these xanthans in 10^?4M NH₄Ac (the disordered conformation) display a mixture of species ranging from unaggregated single- or perfectly matched double-stranded species, to double-stranded chains branching into its two subunits as well as double-stranded chains with different degrees of mismatching. This study suggests that the perfectly matched antiparallel or parallel double-stranded chain constitutes the lowest free energy state of the ordered conformation of xanthan in dilute aqueous solution.     

Interactive measurement and characterization of DNA molecules by analysis of AFM images.

BACKGROUND: In the past few years, computer-based analysis of atomic-force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods. METHODS: Two kinds of images were analyzed: computer-generated filaments that modeled circular DNA molecules on a surface and real atomic-force microscopic images of DNA molecules adsorbed on an appropriate substrate surface. RESULTS: The algorithm was tested on a group of 140 simulated and 189 real plasmids with a nominal length of 913 nm. The accuracy of the length measurement was statistically evaluated on the ensemble of molecules, with particular attention to the influence of the noise. Mean contour lengths of 912 +/- 5 nm and 910 +/- 47 nm were found for simulated and real plasmids, respectively. The measured end-to-end distance of lambda-DNA molecules as a function of their contour length is reported, from which it is possible to estimate the stiffness of the DNA molecules adsorbed onto a surface; the value obtained for the DNA persistence length (42 +/- 5 nm) is consistent with values measured by other imaging techniques. CONCLUSIONS: An interactive algorithm for DNA molecule measurements based on the detection of the filament ridge line in a digitized image is presented. The simulation of artificial filaments combined with the experimental data demonstrates that the proposed method can be a valuable tool for the DNA contour length evaluation, especially in the case of complex shapes where the use of automatic methods is not possible.     

Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment.

We have developed computational techniques that allow image averaging to be applied to electron micrographs of filamentous molecules that exhibit tight and variable curvature. These techniques, which involve straightening by cubic-spline interpolation, image classification, and statistical analysis of the molecules' curvature properties, have been applied to purified brain clathrin. This trimeric filamentous protein polymerizes, both in vivo and in vitro, into a wide range of polyhedral structures. Contrasted by low-angle rotary shadowing, dissociated clathrin molecules appear as distinctive three-legged structures, called "triskelions" (E. Ungewickell and D. Branton (1981) Nature 289, 420). We find triskelion legs to vary from 35 to 62 nm in total length, according to an approximately bell-shaped distribution (mu = 51.6 nm). Peaks in averaged curvature profiles mark hinges or sites of enhanced flexibility. Such profiles, calculated for each length class, show that triskelion legs are flexible over their entire lengths. However, three curvature peaks are observed in every case: their locations define a proximal segment of systematically increasing length (14.0-19.0 nm), a mid-segment of fixed length (approximately 12 nm), and a rather variable end-segment (11.6-19.5 nm), terminating in a hinge just before the globular terminal domain (approximately 7.3 nm diameter). Thus, two major factors contribute to the overall variability in leg length: (1) stretching of the proximal segment and (2) stretching of the end-segment and/or scrolling of the terminal domain. The observed elasticity of the proximal segment may reflect phosphorylation of the clathrin light chains.     

DNA length and concentration dependencies of anisotropic phase transitions of DNA solutions

Critical concentrations for the isotropic to cholesteric phase transitions of double-stranded DNA fragments in simple buffered saline (0.1 M NaCl) solutions were determined as a function of DNA contour length ranging from approximately 50 nm to 2700 nm, by solid-state 31P NMR spectroscopy and polarized light microscopy. As expected for semirigid chains, the critical concentrations decrease sharply with increasing DNA length near the persistence length in the range from 50 to 110 nm, and approach a plateau when the contour length exceeds 190 nm. The biphasic region is substantially wider than observed for xanthan, another semirigid polyelectrolyte approximately twice as stiff as DNA, primarily because of low critical concentrations for first appearance of the anisotropic phase, C(i)*, in DNA samples > or =110 nm (320 base pairs) long. The limiting C(i)* for DNA > or =490 nm long is exceptionally low (only 13 mg/ml) and is substantially lower than the C(i)* of approximately 40 mg/ml reported for the stiffer xanthan polyelectrolyte. The much higher values of the critical concentrations, C(a)*, for the disappearance of the isotropic DNA phase (> or =67 mg/ml) are modestly higher than those observed for xanthan and are predicted reasonably well by a theory that has been applied to other semirigid polymers, if a DNA persistence length in the consensus range of 50-100 nm is assumed. By contrast, the broad biphasic region and low C(i)* values of DNA fragments > or =190 nm long could only be reconciled with theory by assuming persistence lengths of 200-400 nm. The latter discrepancies are presumed to reflect some combination of deficiencies in current theory as applied to chiral, strong polyelectrolytes such as DNA, and sequence-dependent variations in DNA properties such as flexibility, curvature, or interaction potential. The propensity of DNA to spontaneously self-order at low concentrations well in the physiological range may have biological significance.     

Tapping mode atomic force microscopy of scleroglucan networks

Tapping mode Atomic Force Microscopy (TmAFM) has been used to study the fungal polysaccharide scleroglucan deposited from aqueous solution and dimethyl sulfoxide (DMSO) onto a mica surface. The solutions from which the microscope samples were produced were prepared by first dissolving the solid scleroglucan in 0.1M NaOH, then neutralizing the solution with HCl, followed by dilution to the required concentration in either water or DMSO. It was found that from the aqueous solution described above, scleroglucan forms networks. Based on a comparison of the denatured-renatured and aqueous solution samples, network formation is due to the imperfect registration between the chains forming the triple helices. The relatively large stiffness of the scleroglucan triple helix is also assumed to contribute to the formation of the extended networks. The triple helix diameter was measured to be 0.92 ± 0.27 nm, which is in the same range as that obtained by other researchers using similar techniques. Denatured scleroglucan, deposited from DMSO onto mica, forms a web-like layer on top of which there are sphere-like structures. These morphologies are believed to be due to triple helix denaturation yielding highly flexible single chains in DMSO, which results in coiling and web-like dense packing of scleroglucan upon deposition onto mica. Most interestingly after addition of water to the samples deposited from DMSO, some of the chains can be renatured into short, stiff rod-like structures which are similar to the structures observed by other researchers. The imaging data for aqueous solution deposition can be analyzed by plotting maximum end-to-end distance versus the perimeter of the networks deposited onto mica. This yields a Flory-like exponent of 0.67, which is almost similar in value to that obtained by other researchers for linear structures of scleroglucan but less than that expected for a polymer chain following a self-avoiding walk (v = 0.75) model on a two-dimensional surface. The fractal dimension that can be used to characterize the networks was determined graphically to be 1.22 ± 0.06. © 1997 John Wiley & Sons, Inc. Biopoly 42: 89–100, 1997     

Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain

We have studied the structure of bovine heart mitochondrial NADH:ubiquinone (Q) oxidoreductase (EC 1.6.99.3) by image analysis of electron micrographs. A three-dimensional reconstruction was calculated from a tilt-series of a two-dimensional crystal of the molecule. Our interpretation of the position of the molecule in the unit cell of the crystal is supported by additional (low-resolution) analysis of images of single molecules. The three-dimensional reconstruction was calculated with the aid of an iterative real-space reconstruction algorithm. The various projections used as input to the algorithm were obtained by averaging the images of the tilted crystal through a Fourier-space peak-filtering procedure. The reconstructed unit cell measures 15.2 X 15.2 nm in the plane of the two-dimensional crystal and has a height of 10-11 nm. The unit cell contains one molecule consisting of four large subunits. At the present resolution of about 1.3 nm in the untilted projection, these four monomers are seen as two dimers related by a two-fold axis. Two views of the single particles have been recognized; they are the top and side view of the building block of the crystal. After computer image alignment and correspondence analysis, clusters of similar particles have been averaged. In the averages an uneven stain distribution is seen around the molecules, which may result from preferential staining of hydrophilic parts of the molecule. The molecular mass of the whole molecule was determined from scanning transmission electron microscopy measurements as (1.6 +/- 0.2) X 10(6) daltons.     

The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins)

The knowledge accumulated over the last 30 years concerning the subunit structures of the invertebrate extracellular hemoglobins permits us to classify them into four distinct groups. Single-domain, single-subunit hemoglobins consisting of single, heme-binding polypeptide chains which have a molecular mass of ca. 16 KDa. These molecules are found in multicellular parasitic organisms such as the trematodes Dicrocoelium and Fasciolopsis and in a few insects, namely in the adult Anisops and in the larvae of Chironomus and of Buenoa. Two-domain, multi-subunit hemoglobins consisting of 30-37 KDa polypeptide chains each containing two, linearly connected heme-binding domains, which form polymeric aggregates with molecular masses ranging from 250 to 800 KDa. These hemoglobins are found extensively among the carapaced branchiopod crustaceans: Caenestheria, Daphnia and Lepidurus hemoglobins have been found to consist of 10, 16 and 24 two-domain chains, respectively. Judging from their electron microscopic appearances, some of the hemoglobins may possess different molecular symmetries. Multi-domain, multi-subunit hemoglobins consisting of two or more polypeptide chains, each comprising many heme-binding domains of ca. 15-20 KDa each. Examples of this class are found among the carapaceless branchiopod crustaceans, the planorbid snails and the clams from the families Astartidae and Carditidae. Artemia hemoglobin consists of two chains of ca. 125 KDa, each containing 8 heme-binding domains. Planorbis and Helisoma hemoglobins possess a molecular mass of ca. 1700 KDa and consist of 10 chains of 170-200 KDa. Astarte and Cardita hemoglobins appear in electron micrographs as rod-like polymers of variable dimensions, 20-30 nm in diameter and 20-100 nm in length and consist of polypeptide chains of ca. 300 KDa. The crustacean and gastropod hemoglobins vary in their electron microscopic appearance and may possess different molecular symmetries. Single-domain, multi-subunit hemoglobins consisting of aggregates of several small subunits, some of which are disulfide-bonded and not all of which contain heme. These molecules are widely distributed among the annelids and possibly also among the pogonophores. They are characterized by a two-tiered, hexagonal electron microscopic appearance, with a vertex-to-vertex diameter of 30 nm and a height of 20 nm, an acidic isoelectric point, a sedimentation coefficient of 50-60 S and a low iron content of 0.24 +/- 0.03%.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray crystallographic and biochemical characterization of single crystals formed by proteolytically modified human fibrinogen

Large single crystals (0.7 mm X 0.4 mm X 0.3 mm) of human fibrinogen, modified with a crude exoprotease from Pseudomonas aeruginosa, have been obtained. The crystals are orthorhombic, space group P212121, with a = 9.5 +/- 0.1 nm, b = 11.1 +/- 0.1 nm, c = 44.0 +/- 0.4 nm. Their X-ray diffraction patterns extend to beyond 1.0 nm resolution. The asymmetric unit contains one fragment of 245 kDa molecular mass made up of an intact gamma chain, a slightly shortened beta chain and an N-terminal part (about one-third) of the alpha chain. In electron micrographs of rotary-shadowed samples the crystallized particles are very similar in size and shape to the well-known trinodular form of native fibrinogen. From the unit-cell dimensions and the intensity pattern a model is proposed in which the molecules consist of two halves related by a local twofold rotation axis, and are aligned with a displacement of multiples of 1/4 of their length giving a pseudohexagonal packing scheme.     

Native conformation of human von Willebrand protein. Analysis by electron microscopy and quasi-elastic light scattering

von Willebrand factor (vWF) was analyzed by electron microscopic and quasi-elastic light scattering techniques in order to evaluate the size and shape of this heterogeneous polymeric plasma glycoprotein. Electron micrographs demonstrated that native vWF molecules are flexible, linear polymers, ranging in contour length from 100 to 1300 nm. In their typical configuration, the polymers were coiled upon themselves with maximal diameters ranging from 60 to 200 nm. Individual repeating protomeric subunits were discernible in occasionally noted, uncoiled polymers and measured 100 nm X 1.5-2.0 nm. Quasi-elastic light scattering analysis confirmed that measurements of the size and shape of purified vWF molecules in solution were similar to those obtained with electron microscopic techniques. In addition, the mean Stokes radius and mean radius of gyration assessed by quasielastic light scattering were directly related over a wide range of values, as were the diameter and contour length measured from electron micrographs, suggesting that the overall shape of polymers does not change with increasing size. This study supports the concept that native vWF molecules are flexible, linear polymers. In addition, this study clearly shows that the polymer configuration assessed from electron micrographs is a valid representation of the configuration of the polymer in solution. The data presented also provide the first evidence for a well-defined, repeating protomeric subunit.     

Rheological Study of the Gelation Process of Agarose-Based Solutions

The gelation of agarose is investigated by rheological methods and electron microscopy, as well as the thickening properties of xanthan. The gelling and thickening agents have been investigated in pure water to compare the results with theoretical models. The gelation of agarose was shown to follow two steps upon cooling, which could be addressed to the formation of helices and their aggregation. In addition to the rheology, transmission electron micrographs of freeze-dried samples have been taken to underline the date by corresponding structures at different stages of the gelling process. The xanthan molecules, which have been approximated by rigid highly charged rodlike molecules, undergo a jamming transition at a critical concentration. This concentration shows a strong dependence on the length of the molecules, which supports the high thickening effect of xanthan. When both, agarose and xanthan are mixed, the gel structure becomes very different. The gelling process is now determined by one step only. It is proposed that the jamming xanthan molecules prevent the formation of the aggregates of the agarose gel. The gels themselves appear then less elastic, and should yield a better mouth feeling.     

Surface topography and molecular stoichiometry of the mitochondrial channel, VDAC, in crystalline arrays.

The mitochondrial outer membrane contains a protein, called VDAC, that forms large aqueous pores. In Neurospora crassa outer membranes, VDAC forms two-dimensional crystalline arrays whose size and frequency can be greatly augmented by lipase treatment of these membranes (C. Mannella, Science 224, 165, 1984). Fourier filtration and surface reconstruction of freeze-dried/shadowed (45 degrees) arrays produced detailed images of two populations of crystals, whose lattices are mirror images of each other. Most likely, this technique has revealed both surfaces of the same two-dimensional crystal with lattice parameters: a = 12.3 +/- 0.1 nm, b = 11.2 +/- 0.1 nm, and theta = 109 +/- 1 degree. Three-dimensional reconstructions of the surface reliefs on both sides of the crystal show them to be very similar. The majority of the protein forming the channel appears to be at or below the level of the membrane. To address the issue of the number of 30-kDa polypeptides that form a VDAC channel, measurements of mass per unit area were carried out by analyzing scanning transmission electron micrographs of unstained, freeze-dried arrays. The crystal form used for mass analysis contained the same motif of six stain-accumulating centers per unit cell, with p2 symmetry as in the oblique configuration, but it had a different orientation relative to the lattice lines. These data yielded a surface density of 1.9 +/- 0.2 kDa/nm2, indicating that there is a one-to-one ratio between VDAC polypeptides and the channels visualized in filtered electron micrographs, and that VDAC membrane crystals contain 68% protein and 32% lipid by mass.     

Double-stranded helix of xanthan: Rigidity in 0.01M aqueous sodium chloride containing 0.01 N hydrochloric acid

Six samples of Na xanthan in 0.01M aqueous NaCl containing 0.01 N HCl (pH = 2) were studied by light scattering and viscosity. This study was motivated by the finding that the intrinsic viscosity [η] fairly sharply decreased when the pH of the solvent was lowered from about 6 to 2 by adding HCl to 0.01M aqueous NaCl in which Na xanthan dissolves as rigid dimers having a double-helical structure. The data for weight-average molecular weight, radius of gyration, and [η] showed that Na xanthan at pH = 2 remains a dimer behaving as a semiflexible chain. Data analysis in terms of known theories for unperturbed wormlike chains yielded 0.47 ± 0.02, 2.0 ± 0.6, and 68 ± 7 nm for the contour length h per main-chain residue, diameter d, and persistence length q of the dimer, respectively. these h and d values agreed with the pitch per main-chain residue and the diameter of the double helix of Na xanthan in 0.01 or 0.1M aqueous NaCl. However, the q value, which was close to the intrinsic persistence length q₀ ( = q in the absence of electrostatic interaction) of Na xanthan at pH = 2, was much smaller than the q₀ (106 nm) of this helix. We concluded that the xanthan dimer at pH = 2 assumes a double-helical structure, which is geometrically the same as, but is more flexible than, that at neutral pH.     

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.     

Native mitochondrial creatine kinase forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurements by scanning transmission electron microscopy, and image analysis of single mitochondrial creatine kinase octamers

Electron micrographs of negatively stained and metal-shadowed mitochondrial creatine kinase (Mi-CK) molecules purified as described by Schlegel et al. (Schlegel, J., Zurbriggen, B., Wegmann, E., Wyss, M., Eppenberger, H. M., and Wallimann, T. (1988) J. Biol Chem. 263, 16942-16953) revealed a homogeneous population (greater than or equal to 95%) of distinctly sized square-shaped, octameric particles with a side length of 10 nm that frequently exhibited a pronounced 4-fold axis of symmetry. The cube-like molecules consist of four dimers that are arranged around a stain-accumulating central cavity of 2.5-3 nm in diameter. This interpretation is supported by single particle averaging including correlation analysis by computer. Upon prolonged storage or high dilution, the cube-like octamers tended to dissociate into "banana-shaped" dimers. Sedimentation velocity and sedimentation equilibrium experiments yielded an s value of 12.8-13.5 S and an Mr of 328,000 +/- 25,000 for the octameric cubes. An s value of 5.0 S and a Mr of 83,000 +/- 8,000 was found under conditions which revealed banana-shaped dimers. These dimers proved to be very stable, as their dissociation into monomers of 45 kDa (s value = 2.0 S) required 6 M guanidine HCl. Thus, the oligomeric structures observed in the electron microscope are identified as Mi-CK dimers (banana-shaped structures) and cubical Mi-CK octamers assembled from four Mi-CK dimers. The octameric nature of native Mi-CK and the formation of Mi-CK dimers were confirmed by direct mass measurements of individual molecules by scanning transmission electron microscopy yielding a molecular mass of 340 +/- 55 kDa for the octamer and 89 +/- 27 kDa for the dimer. A structural model of Mi-CK octamers and the possible interaction with ATP/ADP-translocator molecules as well as with the outer mitochondrial membrane is proposed. The implications with respect to the physiological function of Mi-CK as an energy-channeling molecule at the producing side of the phosphoryl creatine shuttle are discussed.     

The spindle pole body of the pathogenic yeast Exophiala dermatitidis: variation in morphology and positional relationship to the nucleolus and the bud in interphase cells

The spindle pole body (SPB) in the interphase cell of the pathogenic yeast Exophiala dermatitidis was studied in detail. The SPB was located on the outer nuclear envelope and was 342 +/- 86 nm long in a haploid strain. It consisted of two disk elements that measured 151 +/- 43 nm in diameter and 103 +/- 17 nm in thickness, connected by a rod-shaped midpiece that measured 56 +/- 20 nm in length and 37 +/- 9 nm in diameter. There were considerable variations in size and morphology of interphase SPB. Some disk elements appeared spherical but others were more flattened, and there was variation in electron density. A few SPBs did not have the midpiece. The SPB of a diploid strain was 486 +/- 118 nm long, thus significantly bigger than that of the haploid strain. The SPB tended to be localized away from the nucleolus (110 +/- 48 degrees), but close to the bud (78 +/- 45 degrees). The present study highlights the necessity of observing a large number of micrographs in three-dimensions to describe accurately the ultrastructure of the SPB in yeast.     

Single-molecule measurements of the persistence length of double-stranded RNA

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

Chromatin fibers are left-handed double helices with diameter and mass per unit length that depend on linker length

Four classes of models have been proposed for the internal structure of eukaryotic chromosome fibers--the solenoid, twisted-ribbon, crossed-linker, and superbead models. We have collected electron image and x-ray scattering data from nuclei, and isolated chromatin fibers of seven different tissues to distinguish between these models. The fiber diameters are related to the linker lengths by the equation: D(N) = 19.3 + 0.23 N, where D(N) is the external diameter (nm) and N is the linker length (base pairs). The number of nucleosomes per unit length of the fibers is also related to linker length. Detailed studies were done on the highly regular chromatin from erythrocytes of Necturus (mud puppy) and sperm of Thyone (sea cucumber). Necturus chromatin fibers (N = 48 bp) have diameters of 31 nm and have 7.5 +/- 1 nucleosomes per 10 nm along the axis. Thyone chromatin fibers (N = 87 bp) have diameters of 39 nm and have 12 +/- 2 nucleosomes per 10 nm along the axis. Fourier transforms of electron micrographs of Necturus fibers showed left-handed helical symmetry with a pitch of 25.8 +/- 0.8 nm and pitch angle of 32 +/- 3 degrees, consistent with a double helix. Comparable conclusions were drawn from the Thyone data. The data do not support the solenoid, twisted-ribbon, or supranucleosomal particle models. The data do support two crossed-linker models having left-handed double-helical symmetry and conserved nucleosome interactions.     

Effects of denaturants on the dynamics of loop formation in polypeptides

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.