Imaging of individual biopolymers and supramolecular assemblies using noncontact atomic force microscopy

A variety of biopolymers is imaged using noncontact atomic force microscopy. Samples are prepared by aerosol spray deposition of aqueous solutions on freshly cleaved mica followed by air drying. The distributions of contour lengths and chain or fibril thicknesses normal to the mica substrate can be measured for individual polymer molecules or molecular assemblies. In many cases it is possible to conclude that the structures imaged and quantitatively analyzed are representative of those present in solution and not artifacts of the deposition/dessication process. Imaging of linear and cyclic triple helices of the polysaccharide scleroglucan is demonstrated. Measurements of the triple helix thickness normal to the mica surface are analyzed, and successful measurements of the molecular weight distribution and mean molar mass are described. It is demonstrated that the extent of chain association in the polysaccharide xanthan can be modulated by the addition of low molecular weight salts. The contour length and chain thickness distributions in a xanthan fraction are presented. Increases in the extent of chain association with increasing polymer concentration are documented for the gelling polysaccharide gellan, and the formation of stiff fibrillar gellan aggregates in the presence of added low molecular salt is demonstrated. Images are presented of the polysaccharide κ-carrageenan in its disordered, and presumably single-stranded, state. Biopolymers other than polysaccharides can be imaged by the same technique; this is demonstrated with the fibrous protein collagen. In general it is shown that aerosol spray deposition of biopolymer samples can be used in conjunction with noncontact atomic force microscopy to provide a fast, reliable, and reproducible method for assessing the size and shape distributions of individual biological macromolecules and macromolecular assemblies in solution with a minimum of time and effort devoted to sample preparation. © 1997 John Wiley & Sons, Inc. Biopoly 42: 133–146, 1997     

Direct observation of biopolymers produced from submerged culture of edible mushrooms by atomic force microscopy

Two biopolymers produced from submerged culture of edible mushrooms were directly observed by atomic force microscopy. Biopolymers were deposited on mica from dilute aqueous solution and imaged in air through a thin layer of adsorbed water and their hydrated structures were observed by a tapping mode. A single biopolymer molecule obtained from Cordyceps militaris was typical of a rod-like structure with bending point, which can form intra- and inter-molecular supercoils. In contrast, the image for low molecular weight biopolymer from Paecilomyces sinclarii is typical of a branched structure in which more extensive interaction leads to the formation of network-like matrix.     

Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains.

The extracellular matrix polysaccharide hyaluronan has been examined by tapping mode atomic force microscopy. High molecular weight hyaluronan was deposited on mica from dilute aqueous solution and imaged in air. Long unbranched chains could be observed and were found to be compatible with the known covalent structure of hyaluronan. In addition, chains with evidence of intramolecular association were observed. In the simplest cases, the association took the form of loops stabilized by antiparallel double-stranded (probably double-helical) segments. In other cases, the polarity of the associated regions could not be determined. Extensive intramolecular association in long hyaluronan chains resulted in a fenestrated structure of the same type as that formed by intermolecular association at higher concentrations.     

Interaction of the alternating double stranded copolymer poly(dA-dT) x poly(dA-dT) with NiCl(2) and CdCl(2): solution behavior

The thermal denaturation of the synthetic high molecular weight double stranded polynucleotide poly(dA-dT) x poly(dA-dT) has been studied in aqueous buffered solution (Tris 1.0 mM; pH 7.8+/-0.2) in the presence of increasing concentrations of either Ni(2+) (borderline cation) or Cd(2+) (soft cation) at four different constant ionic strength values (NaCl), making use of UV and circular dichroism (CD) spectroscopies. The experimental results show that the B-type double helix of the polymer is stabilized against thermal denaturation in the presence of both cations at low concentrations, relative to the systems where only NaCl is present, in the same conditions of ionic strength and pH. The effect is more pronounced for Ni(2+) than for Cd(2+). At higher concentrations, both cations start to destabilize the double helix, with Cd cations inducing larger variations of T(m). In many cases, when denaturation starts, interstrand cross-linking occurs with formation of aggregates that precipitate.     

Circular dichroism of polynucleotides: Interactions of NiCl2 with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) in a water-in-oil microemulsion

The thermal behavior of the synthetic, high molecular weight, double stranded polynucleotides poly(dA-dT).poly(dA-dT) [polyAT] and poly(dG-dC).poly(dG-dC) [polyGC] solubilized in the aqueous core of the quaternary water-in-oil cationic microemulsion CTAB|n-pentanol|n-hexane|water in the presence of increasing amounts of NiCl(2) at several constant ionic strength values (NaCl) has been studied by means of circular dichroism and electronic absorption spectroscopies. In the microemulsive medium, both polynucleotides show temperature-induced modifications that markedly vary with both Ni(II) concentration and ionic strength. An increase of temperature causes denaturation of the polyAT duplex at low nickel concentrations, while more complex CD spectral modifications are observed at higher nickel concentrations and ionic strengths. By contrast, thermal denaturation is never observed for polyGC. At low Ni(II) concentrations, the increase of temperature induces conformational transitions from B-DNA to Z-DNA form, or, more precisely, to left-handed helical structures. In some cases, at higher nickel concentrations, the CD spectra suggest the presence of Z'-type forms of the polynucleotide.     

The presence of RNA in a double helix inhibits its interaction with histone protein

The binding of core histones (H2A, H2B, H3, H4) to a circular plasmid DNA and to a circular DNA-RNA hybrid molecule of similar size has been compared. Circular hybrid molecules were formed from single stranded fd DNA by synthesis of the complimentary strand with ribonucleotides using wheat germ RNA polymerase II. Upon reconstitution of plasmid DNA circles with histone, the sedimentation profiles of the DNA remained sharp by increased several fold in rate. Material from the peak fractions of these sedimentations appeared to be condensed circular loops of nucleosomes when examined by electron microscopy (EM), and the mass ratio of DNA to histone (at the histone concentrations which produced the fastest sedimentations) was typical of native chromatin. In contrast, the sedimentation behavior of DNA-RNA hybrid circles after addition of histone remained unchanged except for a minor fraction which exhibited a broad and faster sedimentation rate. Examination by EM revealed that most of the molecules appeared identical to protein free hybrid circles while the minor, faster sedimenting fraction appeared to be two or more circles bound together by protein aggregates. Finally, a linear molecule consisting of about 3000 base pairs of duplex DNA covalently joined on both ends to 1500 base pairs of RNA-DNA hybrid helix was constructed. Reconstitution of this molecule with core histone showed nucleosome formation only on the central DNA duplex region. Isopycnic banding of fixed hybrid-histone mixtures showed that little or no histone had bound to the bulk of the full hybrid molecules. We suggest that the presence of RNA in a nucleic acid duplex inhibits the condensation of the duplex into a nucleosomal structure by histone.     

The binding of RecA protein to duplex DNA molecules is directional and is promoted by a single stranded region.

RecA protein from E. coli binds more strongly to single stranded DNA than to duplex molecules. Using duplex DNA that contains single stranded gaps, we have studied the protection by RecA protein at various concentrations, of restriction sites as a function of their distance from the single stranded region. We show that the binding of RecA protein, initiated in the single stranded region, extends progressively along the adjoining duplex in the 5' to 3' direction with respect to the single stranded region. The strand exchange reaction is known to proceed in the same direction.     

Determination of molecular size and shape of hyperbranched polysaccharide in solution

The chemical structure of a water-soluble polysaccharide, coded as TM3b, extracted from sclerotia of Pleurotus tuber-rigium was analyzed to be a hyperbranched beta-D-glucan with beta-(1-->6), beta-(1-->4), and beta-(1-->3)-linked residues, with degree of branching (DB) of 57.6%. The results from size-exclusion chromatography combined with laser light scattering (SEC-LLS) revealed that the hyperbranched polysaccharide easily aggregated in 0.15 M aqueous NaCl, whereas it dispersed as individual chains in DMSO. The weight-average molecular weight (M(w)), radius of gyration, intrinsic viscosity, and chain density of TM3b in DMSO and in 0.15 M aqueous NaCl were measured with SEC-LLS, LLS, and viscometry. The results indicated that single chains and aggregates with aggregation number of 12 coexisted in the aqueous solution, whereas individual molecules of TM3b occurred in DMSO. In view of the molecular parameters, the aggregates in aqueous solution exhibited more compact chain structure than the individual molecules in DMSO. Furthermore, transmission electron microscopy and atomic force microscopy showed that all of the aggregates and individual molecules exhibited spherical particles in the solutions. This work provided the valuable information of chain conformation and molecular morphology of the hyperbranched polysaccharide in different solvents.     

Macromolecular properties of cepacian in water and in dimethylsulfoxide

Cepacian is the exopolysaccharide produced by the majority of the so far investigated clinical strains of the Burkholderia cepacia complex. This is a group of nine closely related bacterial species that might cause serious lung infections in cystic fibrosis patients, in some cases leading to death. In this paper the aggregation ability and the conformational properties of cepacian chain were investigated to understand its role in biofilm formation. Viscosity and atomic force microscopy studies in water and in mixed (dimethylsulfoxide/water) solvent indicated the formation of double stranded molecular structures in aqueous solutions. Inter-residue short distances along cepacian chain were investigated by NOE NMR, which showed that two side chains of cepacian were not conformationally free due to strong interactions with the polymer backbone. These interactions were attributed to hydrogen bonding and contributed to structure rigidity.     

Force spectroscopy of the double-tethered concanavalin-A mannose bond

We present the measurement of the force required to rupture a single protein-sugar bond using a methodology that provides selective discrimination between specific and nonspecific binding events and helps verify the presence of a single functional molecule on the atomic force microscopy tip. In particular, the interaction force between a polymer-tethered concanavalin-A protein (ConA) and a similarly tethered mannose carbohydrate was measured as 47 +/- 9 pN at a bond loading rate of approximately 10 nN/s. Computer simulations of the polymer molecular configurations were used to determine the angles that the polymers could sweep out during binding and, in conjunction with mass spectrometry, used to separate the angular effects from the effects due to a distribution of tether lengths. We find that when using commercially available polymer tethers that vary in length from 19 to 29 nm, the angular effects are relatively small and the rupture distributions are dominated by the 10-nm width of the tether length distribution. In all, we show that tethering both a protein and its ligand allows for the determination of the single-molecule bond rupture force with high sensitivity and includes some validation for the presence of a single-tethered functional molecule on the atomic force microscopy tip.     

In Silico Modeling and Conformational Mobility of DNA Duplexes

The review is focused on issues of transferability of the context-sensitive conformational characteristics of DNA estimated from crystallographic structural data on the DNA in aqueous solution. The state of the art in molecular dynamics of charged biopolymers in aqueous solution is covered. Elaboration of expedient force fields and algorithms of calculating long-range electrostatic interactions and solving combined equations of atomic motion have made it possible to generate stable nanosecond trajectories of thermal atomic motion of the biopolymer in aqueous solution in the presence of counterions and salt ions over reasonable time. Tools for analyzing the atomic statistical trajectories of DNA duplexes in aqueous solution to infer context-dependent conformational dynamic characteristics are discussed together with advances in simulating the mechanisms of global axial bend in DNA duplexes. These techniques allow one to consecutively analyze relationships between the contextual composition of the duplex and the basic modes of essential motions, their amplitude and extent of fluctuation. Development of satisfactory methods for estimating the free energy of biopolymer conformations in solution permits qualitative assessment of the conformational thermodynamic stability of biopolymers and their complexes.     

Methods of computer modeling and conformational mobility of DNA duplexes

The review is focused on issues of transferability of the context-sensitive conformational characteristics of DNA estimated from crystallographic structural data on the DNA in aqueous solution. The state of the art in molecular dynamics of charged biopolymers in aqueous solution is covered. Elaboration of expedient force fields and algorithms of calculating long-range electrostatic interactions and solving combined equations of atomic motion have made it possible to generate stable nanosecond trajectories of thermal atomic motion of the biopolymer in aqueous solution in the presence of counterions and salt ions over reasonable time. Tools for analyzing the atomic statistical trajectories of DNA duplexes in aqueous solution to infer context-sensitive conformational dynamic characteristics are discussed together with advances in simulating the mechanisms of global axial bend in DNA duplexes. These techniques allow one to consecutively analyze relationships between the contextual composition of the duplex and the basic modes of essential motions, their amplitude and extent of fluctuation. Development of satisfactory methods for estimating the free energy of biopolymer conformations in solution permits qualitative assessment of the conformational thermodynamic stability of biopolymers and their complexes.     

Rheological and microstructural investigation of oat β-glucan isolates varying in molecular weight

The rheological properties and microstructure of aqueous oat β-glucan solutions varying in molecular weight were investigated. The structural features and molecular weights (MW) were characterized by ¹³C NMR spectroscopy and high performance size-exclusion chromatography (HPSEC), respectively. The microstructure of the β-glucans dispersions was also examined by atomic force microscopy (AFM). The samples with β-glucan content between 78 and 86% on a dry weight basis had MW, intrinsic viscosity ([η]) and critical concentration (c*) in the range of 142-2800 × 10³ g/mol, 1.7-7.2 dl/g and 0.25-1.10 g/dl, respectively. The flow and viscoelastic behaviour was highly dependent on MW and on the concentration of the β-glucans dispersions. Pseudoplastic behaviour was exhibited at high concentrations and Newtonian behaviour was evident at low concentrations. At the same concentration, the viscosity was higher for higher MW samples. The Cox-Merz rule was applicable for the lower molecular weight samples at higher concentrations whereas the high molecular weight sample deviated at concentrations greater than 1.0%, w/v. The mechanical spectra with variation of both MW and concentration were typical of entangled biopolymer solutions. AFM images revealed the formation of clusters or aggregates linked via individual polymer chains scattered heterogeneously throughout the system. The aggregate size increased with the molecular weight of the samples investigated and has been linked to the rheological behaviour of the samples.     

Atomic force microscopy of a hybrid high-molecular-weight glutenin subunit from a transgenic hexaploid wheat

The high-molecular-weight glutenin subunits (HMW-GS) of wheat gluten in their native form are incorporated into an intermolecularly disulfide-linked, polymeric system that gives rise to the elasticity of wheat flour doughs. These protein subunits range in molecular weight from about 70 K-90 K and are made up of small N-terminal and C-terminal domains and a large central domain that consists of repeating sequences rich in glutamine, proline, and glycine. The cysteines involved in forming intra- and intermolecular disulfide bonds are found in, or close to, the N- and C-terminal domains. A model has been proposed in which the repeating sequence domain of the HMW-GS forms a rod-like beta-spiral with length near 50 nm and diameter near 2 nm. We have sought to examine this model by using noncontact atomic force microscopy (NCAFM) to image a hybrid HMW-GS in which the N-terminal domain of subunit Dy10 has replaced the N-terminal domain of subunit Dx5. This hybrid subunit, coded by a transgene overexpressed in transgenic wheat, has the unusual characteristic of forming, in vivo, not only polymeric forms, but also a monomer in which a single disulfide bond links the C-terminal domain to the N-terminal domain, replacing the two intermolecular disulfide bonds normally formed by the corresponding cysteine side chains. No such monomeric subunits have been observed in normal wheat lines, only polymeric forms. NCAFM of the native, unreduced 93 K monomer showed fibrils of varying lengths but a length of about 110 nm was particularly noticeable whereas the reduced form showed rod-like structures with a length of about 300 nm or greater. The 110 nm fibrils may represent the length of the disulfide-linked monomer, in which case they would not be in accord with the beta-spiral model, but would favor a more extended conformation for the polypeptide chain, possibly polyproline II.     

Molecular distribution and charge of polylysine layers at the surface of lipid membranes and mica

Electrophoretic mobility of cardiolipin liposomes was measured in the presence of polylysines of different molecular weight at various concentrations of the background electrolyte (KCl). The electrophoretic mobility in a liposome suspension changes its sign and reaches a plateau at high polylysine content. The surface charge in the plateau region was determined according to the Gouy-Chapman model of the electrical double layer. The average charge density was found to equal 0.005 and 0.016 Coulomb/m² for the polymer length of 5 and 12 units (bases), respectively, and 0.032 Coulomb/m² for polylysines with the length of 130 and 1435 units. The molecular distribution of these polylysines was studied at the mica surface using atomic force microscopy in the 10-mM KCl solution. It was shown that pentalysine molecules covered uniformly about 90% of the surface with the layer thickness of about 0.8 nm. The high-molecular polylysines cover about 60% of the surface with the layer thickness of more than 1.5 nm. The data suggest that the polymer forms a compact layer on the membrane surface; the charge density at the outer surface is determined both by the polymer properties and by the total amount of anionic lipids, irrespective of their ionization state.     

Structure and dynamics of the complex of single stranded DNA binding protein of Escherichia coli with circular single stranded DNA of filamentous phages

We have analyzed the equilibrium and nonequilibrium properties of the complex of the single stranded DNA binding protein of Escherichia coli (EcoSSB) and circular single stranded DNA of filamentous phages M13mp8 and F1 using static and dynamic light scattering, analytical ultracentrifugation and electron microscopy. Upon binding to the single stranded DNA the EcoSSB tetramer replaces an equivalent volume of water trapped within the coiled single stranded DNA and hinders the folding of the single stranded DNA into secondary structures at all salt concentrations. The salt dependent compaction of the stoichiometric complex can be described assuming a flexible polyelectrolyte chain. The solution structure of the macromolecular complex is a random coil and in the electron microscope a beaded flexible structure of the complex with a bead diameter of 6 nm appears at all salt concentrations used. The internal motions of the stoichiometric complex can be described by the Rouse-Zimm model of polymer dynamics. The segmental mobility of the complex can be correlated with changes in the binding site size of the EcoSSB tetramer; it indicates the presence of interactions between EcoSSB tetramers bound to single stranded DNA.     

Atomic force microscopy of supported lipid membranes and their complexes with polycations

The method of atomic force microscopy has been used to investigate the morphology of mica-supported bilayer lipid membranes and stability of their complexes with a cationic polymer, poly-(N-ethyl-4-vinylpyridinium bromide). Lipid bilayers with a minimum of defects were obtained by the fusion of monolamellar neutral or mixed anionic bilayer vesicles (liposomes) on the mica surface, followed by excessive solvent removal by means of rapid rotation of a plate in horizontal plane (spin-coating). It has been shown that the cationic polymer does not interact with the bilayers, where the outer leaflet (i.e., the monolayer exposed to the surrounding aqueous solution) is made of an electroneutral phosphatidylcholine (PC). At the same time, the polymer irreversibly binds to the bilayer containing an anionic lipid.     

Molecular weight and related properties of lily amylose determined by monitoring of elution from TSK-GEL PW high performance gel chromatography columns by the low-angle laser light scattering technique and precision differential refractometry

Amylose was fractionated according to its molecular weight by high performance gel chromatography using columns of a TSK-GEL PW series. Elution from the columns was monitored with a low-angle laser light scattering photometer and a precision differential refractometer. The following results were obtained indicating that the procedure is highly efficient for characterizing an amylose preparation with respect to its molecular weight: 1) the weight-average molecular weight of lily amylose used as a test material was determined to be 786,000 +/- 26,000 (n = 7); 2) the molecular weight distribution curve of the amylose was worked out from the chromatographic data; and 3) based on the concept of the universal calibration curve, the Mark-Houwink-Sakurada equation of the amylose was presumed to be [eta] = 2.27 X 10(-4)M0.62 (dl/g). The technique saves time and sample significantly compared with the conventional ones, and consequently enables the characterization of amylose in aqueous solvents without either the degradation or association peculiar to the amylose molecule.     

Directly investigating the interaction between aptamers and thrombin by atomic force microscopy

Aptamers are single‐stranded nucleic acid molecules that can be used for protein recognition, detection, and inhibition. Over the past decades, two thrombin‐binding aptamers (15apt and 27apt) were reported by systemic evolution of ligands by exponential enrichment technique. Though many studies have been reported about the interactions between the aptamers and thrombin by atomic force microscopy, the thrombins in those studies were all immobilized by chemical agents. Recently, we developed a new method using atomic force microscopy to directly investigate the specific interactions between thrombin and its two aptamers without immobilizing the thrombin. Furthermore, the unbinding dynamics and dissociation energy landscapes of aptamer/thrombin were discussed. The results indicate that the underlying interaction mechanisms of thrombin with its two aptamers will be similar despite that the structures of 15apt and 27apt are different in buffer solution. Copyright © 2013 John Wiley & Sons, Ltd.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.