Single Macromolecule Research Articles

The effect of local charge reactivity modeled with a single large polymer macromolecule : Kroo, E. Preprints of Symposia — American Chemical Society, Division of Fuel Chemistry, 2002, 47, (2), 583–587

The effect of local charge reactivity modeled with a single large polymer macromolecule : Kroo, E. Preprints of Symposia — American Chemical Society, Division of Fuel Chemistry, 2002, 47, (2), 583–587

Architecture and solution properties of amphiphilic polymer brushes with peripheral charged ions

Functionalized poly(ethylene oxide) (PEO) macromonomers ( α-tertiary amino and ω-methacryloyl groups) were prepared by ring-opening polymerization of live PEO anions end-capped with styrene oxide using 2-[2-( N, N-dimethylamino)-ethoxy]ethanol potassium alkoxides as an initiator with methacryloyl chloride. PEO brushes were synthesized by free-radical homopolymerization of such PEO macromonomers. These brushes were converted into peripherally charged brushes by quaternization. We studied the solution properties of both types of brushes from the viewpoint of charge effect. It was found from dynamic light scattering (DLS) that the polymer brushes formed a single macromolecule in solution due to crowding of side chains. It was speculated from angular dependence measurements that the polymer brushes with large aspect ratios took a geometrical anisotropic conformation such as a cylinder. In methanol with a low dielectric constant, radius of gyration ( R G ), and cross-sectional radius of gyration ( R G,C ) of the polymer brushes with charged side chains were smaller than those of the polymer brushes without charges. In a solvent with a low dielectric constant such as methanol, ionic groups do not dissociate and condense. On the other hand, these physical values in an aqueous solution were somewhat larger than those of the polymer brushes without charges. In water with a high dielectric constant, peripherally charged brushes were strongly stabilized by electric double layers.

Experimental lensless soft-X-ray imaging using iterative algorithms: phasing diffuse scattering.

Images of randomly placed two-dimensional arrays of gold balls have been reconstructed from their soft-X-ray transmission diffraction patterns. An iterative hybrid input-output (HiO) algorithm was used to solve the phase problem for the continuous distribution of diffuse X-ray scattering. Knowledge of the approximate size of the clusters was required. The images compare well with scanning electron microscope (SEM) images of the same sample. The use of micrometre-sized silicon nitride window supports is suggested, and absorption filters have been used to allow collection of low spatial frequencies often obscured by a beam stop. This method of phasing diffuse scattering may have application to scattering from individual inorganic nanostructures or single macromolecules.

Chain length dependence of the state diagram of a single stiff-chain macromolecule: Theory and Monte Carlo simulation

We present a Monte Carlo computer simulation and theoretical results for the dependence of the state diagram of a single semiflexible chain on the chain length. The calculated transition lines between different structures in the state diagrams for both studied chain lengths N=40 and N=80 can be described by theoretical predictions which include chain length dependence explicitly. The stability criteria of different structures are discussed. The theoretically predicted exponent in the dependence of the toroid size on the chain length is compatible with computer simulation results.

Combined optical trapping and single-molecule fluorescence

BackgroundTwo of the mainstay techniques in single-molecule research are optical trapping and single-molecule fluorescence. Previous attempts to combine these techniques in a single experiment – and on a single macromolecule of interest – have met with little success, because the light intensity within an optical trap is more than ten orders of magnitude greater than the light emitted by a single fluorophore. Instead, the two techniques have been employed sequentially, or spatially separated by distances of several micrometers within the sample, imposing experimental restrictions that limit the utility of the combined method. Here, we report the development of an instrument capable of true, simultaneous, spatially coincident optical trapping and single-molecule fluorescence.ResultsWe demonstrate the capability of the apparatus by studying force-induced strand separation of a rhodamine-labeled, 15 base-pair segment of double-stranded DNA, with force applied perpendicular to the axis of the DNA molecule. As expected, we observed abrupt mechanical transitions corresponding to the unzipping of DNA at a critical force. Transitions occurred concomitant with changes in the fluorescence of dyes attached at the duplex ends, which became unquenched upon strand separation.ConclusionsThrough careful optical design, the use of high-performance spectral notch filters, a judicious choice of fluorophores, and the rapid acquisition of data gained by computer-automating the experiment, it is possible to perform combined optical trapping and single-molecule fluorescence. This opens the door to many types of experiment that employ optical traps to supply controlled external loads while fluorescent molecules report concurrent information about macromolecular structure.

Encapsulation of Polypyrrole by Internal Domain Modification of Double-Cylinder-Type Copolymer Brushes

Double-cylinder-type copolymer brushes were synthesized by free-radical polymerization of vinylbenzyl-terminated poly(α-methylstyrene)-block-poly(2-vinylpyridine) (PMS-block-P2VP) diblock macromonomers. These copolymer brushes formed a single macromolecule even in good solvent due to crowded side chains. To design the encapsulation of conducting polymers, Cu2+ ions (oxidant) were introduced internal P2VP cylindrical domains by complexation based on the pyridine nitrogen interacting with metal ions. Encapsulation of conducting polymers was performed by the oxidation polymerization of double-cylinder-type copolymer brushes complexed with such an oxidizing agent being added to a mixture of pyrrole and water vapor. It was found from light scattering data that the encapsulation materials of polypyrrole (P2VP block 8 mol %) obtained after chemical modifications formed a single macromolecule in good solvent such as benzene.

Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography

Electron tomography of vitrified cells is a noninvasive three-dimensional imaging technique that opens up new vistas for exploring the supramolecular organization of the cytoplasm. We applied this technique to Dictyostelium cells, focusing on the actin cytoskeleton. In actin networks reconstructed without prior removal of membranes or extraction of soluble proteins, the cross-linking of individual microfilaments, their branching angles, and membrane attachment sites can be analyzed. At a resolution of 5 to 6 nanometers, single macromolecules with distinct shapes, such as the 26S proteasome, can be identified in an unperturbed cellular environment.

ChemInform Abstract: Single Isolated Macromolecules at Surfaces.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation.

Recently, near-infrared (NIR) fluorescence light has been applied to image various biological events in vivo, because it penetrates tissue more efficiently than light in the visible spectrum. Compounds exhibiting fluorescent properties in the NIR range are key elements for this upcoming optical imaging technology. In this paper, we report the synthesis of four new, water-soluble NIR cyanine fluorochromes which have superior chemical stability and optical properties. Each fluorochrome was designed with a monoreactive carboxyl group for labeling purposes. When multiple fluorochromes were attached to a single macromolecule, fluorescence quenching was observed. On the basis of this property, a novel autoquenched enzyme sensitive NIR fluorescence probe was prepared.

Fluorescence Probe and Scanning Force Microscopic Studies of Water Soluble Comb-Shaped Copolymers Consisting of a Hydrophobic Poly(p-alkylstyrene) Main Chainand Hydrophilic Poly(ethylene oxide) Grafted Chains

Solution properties of regular amphiphilic comb-shaped polymers have been studied in water by means of fluorescence technique using pyrene as a probe, dynamic light scattering, 1H NMR, and scanning force microscopy (SFM). They consist of a hydrophobic poly(p-alkylstyrene) as a main chain and hydrophilic poly(ethylene oxide) (PEO) chains as a graft chain. Two types of the comb-shaped polymers have been explored, which were prepared by micellar, organized homopolymerization of amphiphilic PEO macromonomers (1) (C1-EOn-(CH2)m-S, with a p-alkylstyrene ((CH2)m-S) end group, with m=1 and 7, and a PEO chain with n=18, 46, 115, and 469) and by copolymerization of C1-EO18-(CH2)7-S with styrene in water, respectively. Partitioning, I1/I3, and excimer formation of pyrene were studied for their dilute aqueous solutions, and rationalized with the local concentration and structure of the comb-shaped polymers. Hydrophobic pyrene molecules were principally dissolved into the vicinity of the hydrophobic p-heptylstyrene main chain of the comb-shaped polymers. The nature of the hydrophobic main chain of poly(styrene-co- C1-EO18-(CH2)7-S) was found to be essentially similar to that of a series of poly(C1-EOn-(CH2)m-S). Direct observation of single macromolecule of poly(C1-EO46-(CH2)7-S) by SFM revealed a high molecular weight, cylindrical brush-like conformation with ca. 12.8 nm in thickness. In contrast, small fine particles were observed for poly(styrene-co-C1-EO18-(CH2)7-S), suggesting the formation of a unimolecular micelle in water.

A novel three-dimensional variant of the watershed transform for segmentation of electron density maps

Electron density maps at moderate resolution are often difficult to interpret due to the lack of recognizable features. This is especially true for electron tomograms that suffer in addition to the resolution limitation from low signal-to-noise ratios. Reliable segmentation of such maps into smaller, manageable units can greatly facilitate interpretation. Here, we present a segmentation approach targeting three-dimensional electron density maps derived by electron microscopy. The approach consists of a novel three-dimensional variant of the immersion-based watershed algorithm. We tested the algorithm on calculated data and applied it to a wide variety of electron density maps ranging from reconstructions of single macromolecules to tomograms of subcellular structures. The results indicate that the algorithm is reliable, efficient, accurate, and applicable to a wide variety of biological problems.

Perspectives on Single Molecule Diffraction Using the X-Ray Free Electron Laser

A free electron laser capable of operating at the short wavelengths and high energies characteristic of hard X-rays, could be a potentially powerful new tool for the determination of the structures of single biological macromolecules, subcellular organelles, or whole cells at high resolution. Current methods using relatively low-intensity, incoherent X-ray sources depend upon the high signal gain achieved by the coherent scattering of a large number of identical molecules from a crystalline sample. The prerequisite crystallization of biological macromolecules is a major bottleneck in this process and the discrete nature of the Fourier transforms of crystalline materials also complicates the process of the reconstruction of the original molecular structure from its corresponding diffraction pattern. The ability to record detailed diffraction patterns from single molecules would eliminate the need for crystalline samples. Furthermore, the continuous molecular transforms of single molecules can be sampled finely enough to enable the unrecorded phase information to be retrieved from the set of structural amplitudes without recourse to the additional, a priori structural information that is generally required for phase assignment using sets of diffraction amplitudes from crystalline samples. Currently, diffraction experiments with single biological macromolecules or other non-crystalline material are hampered by the significant radiation damage to the samples that occurs as a result of the very large radiation doses that are required. Model calculations indicate that this problem may be solved by using the ultra-short (< 100 fs) pulses of hard X-radiation that a new generation of X-ray free electron lasers should be capable of producing. In anticipation that the current, rapid development of this technology will make this potential a reality in the near future, the theoretical foundations of this new methodology are already being laid.

Retrostructural analysis of poly(vinyl chloride) particles

AbstractA poly(vinyl chloride) (PVC) matrix of final particles is investigated by means of quasi‐elastic light scattering (QELS) and high resolution porosimetry (HRP). It is demonstrated that the QELS method is a powerful tool for the detection of various higher organized structures in particle interiors, which in all likelihood will be a difficult‐to‐process fraction of the resin. Measurements of common PVC grades revealed that the QELS records are usually composed of three distinctly separated size distributions, which were assigned to the size distribution of single macromolecules dissolved (maximum at ca. 10 nm), associates of macromolecules (primary particles, the maximum at ca. 100–200 nm), and microglobules (the maximum at ca. 0.8–1 (μm)). The properties of primary particles are presented, as well as the correlation between QELS and HRP records. Based on this correlation, it can be suggested that the pore size distribution is in fact a morphological fingerprint of individual PVC resins. Some consequences of the results obtained for the evaluation of the processsing properties of PVC are discussed.

Equations for Stochastic Macromolecular Mechanics of Single Proteins: Equilibrium Fluctuations, Transient Kinetics, and Nonequilibrium Steady-State

A modeling framework for the internal conformational dynamics and external mechanical movement of single biological macromolecules in aqueous solution at constant temperature is developed. Both the internal dynamics and the external movement are stochastic; the former is represented by a master equation for a set of discrete states, and the latter is described by a continuous Smoluchowski equation. Combining these two equations into one yields a comprehensive theory for the Brownian dynamics and statistical thermodynamics of single macromolecules. This approach is shown to have wide applications. It is applied to protein−ligand dissociation under external force, unfolding of polyglobular proteins under extension, movement along linear tracks of motor proteins against load, and enzyme catalysis by single fluctuating proteins. As a generalization of the classic polymer theory, the dynamic equation is capable of characterizing a single macromolecule in aqueous solution, in probabilistic terms, (1) its thermo...

Entropy production and excess entropy in a nonequilibrium steady-state of single macromolecules.

Based on a recently developed formalism for mesoscopic stochastic dynamics of single macromolecules, such as motor proteins, in aqueous solution, we demonstrate mathematically the principle of the nonequilibrium thermodynamics originated by the Brussels group. The key concepts of excess entropy and excess entropy production, and their mathematical properties as well as physical interpretations, are discussed. The newly developed stochastic macromolecular mechanics is consistent with the general theory of nonequilibrium thermodynamics far from equilibrium, and more importantly, it bridges the abstract theory with the current experimental and modeling work on molecular motors and other biological systems in nonequilibrium steady state.

Mesoscopic nonequilibrium thermodynamics of single macromolecules and dynamic entropy-energy compensation.

We introduce axiomatically a complete thermodynamic formalism for a single macromolecule, either with or without detailed balance, in an isothermal ambient fluid based on its stochastic dynamics. With detailed balance, the theory yields mesoscopic, nonequilibrium for entropy (Upsilon(t)) and free energy (Psi(t)) of the macromolecule. Upsilon(t) and Psi(t) fluctuate. Expectation (d/dt)E[Psi(t)]< or =0, "="holds if and only if the macromolecule is at thermal equilibrium, in which we show that Upsilon(t) still fluctuates but Psi(t) is a constant. The entropy fluctuation of Landau, E[(DeltaUpsilon(t))(2)], precisely matches the fluctuation in the internal energy, which in turn equals the fluctuation in heat dissipation. As a generalization of Clausius' classic result, the dynamic fluctuations in the entropy and energy of the macromolecule are exactly compensated at thermal equilibrium. For systems with detailed balance, Helmholtz free energy is shown to be the potential of Onsager's thermodynamic force.

Probing the Machinery of Intracellular Trafficking with the Atomic Force Microscope

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular-scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor-ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.

Kinetic behaviour of the system composed of nanosized gold particles and very-high-molecular-weight polyacrylamide

We have investigated a process of aggregation of large macromolecules of polyacrylamide (PAAm) and gold particles of nanometre size suspended in aqueous medium. In the presence of PAAm, the resistance of gold particles to the coagulating action of electrolytes increases considerably. A study of the salt-induced coagulation of gold particles proceeding in the presence of PAAm has shown that the initial aggregation of particles and macromolecules begins as a rapid process, which slows down after some time. In the incipient stage, a single macromolecule can attach a number of particles forming a multiplet. If there are free particles left in the system, a subsequent process of particle-multiplet association occurs, as observed in dynamic light scattering experiments. A considerable increase in the relaxation time of the process was interpreted as a result of the multiplet reconformation needed for the attachment of an additional particle. The changes in the multiplet properties were followed spectroscopically. The long-term changes in light extinction spectra were attributed to the rearrangement of particles within multiplets. The behaviour of the system is affected by the presence of simple electrolytes, which reduce the multiplet stability.

Nano-scaled ordering in highly branched regular polymer structures

General features of the structural moieties spatial arrangement were studied by a number of experimental techniques for a representative set of a novel class of dendritic macromolecules: regular polyorganosiloxane dendrimers, polyallylcarbosilane dendrimers with functional groups, polyallylcarbosilane hyperbranched macromolecules, hyperbranched polyethoxysiloxane macromolecules on the way of their transformation towards molecular form of silica, hyperbranched structures based on the products of hydrolysis of Ti(OBu) 4 and prepared by a sol-gel method. Dendrimers in solution seemed like ball-shaped uniform particles with the averaged volume density close to their bulk density, whereas hyperbranched macromolecules had much less value of the averaged density. A rather general multilevel structural hierarchy was revealed and justified for the studied hyperbranched macromolecules of dendritic structure, namely: an ultra-dispersed subparticle - a fractal cluster - a single fractal macromolecule -particle. The peculiarities of a spatial arrangement of the highly branched regular and irregular dendritic macromolecules were tightly related with the results of the molecular dynamic experiments and with the experimental spectra of their molecular mobility. The comparison was made with the data obtained for dendritic macromolecules of other than studied chemical structures and published elsewhere.

Structure and properties of single complex macromolecules and of related bulk systems

Structure and dynamics of complex macromolecules in computer simulated systems is analyzed. The algorithm based on cooperative molecular rearrangements is applied to various macromolecular structures represented in a simplified form on the lattice. Various macromolecular architectures such as linear chains, stars, dendrimers, bottle-brush polymers as well as cyclic chains and catenane are considered both as single molecules and as dense systems corresponding to polymer melts. A broad range of structural parameters characteristic for each system is taken into account. In some cases, the simulation results are compared with the behavior of real systems in which the structure and dynamics has been studied by X-ray scattering and mechanical spectroscopy, respectively.