Session A18 - Biopolymers.
ORAL session, Monday morning, March 12
Room 307-308, Washington State Convention Center

[A18.001] Exploring Heterogeneities in Actin Networks

The technique of microrheology is now widely applied to study the microscopic viscoelastic properties of complex fluids, including polymer networks. The thermal motion of single beads embedded in the polymer is used to extract its local viscoelastic moduli. However, comparison of bulk and micro-rheological results exhibit discrepancies, which are thought to be either due to the heterogeneous nature of the network or the coupling of the probe particles to the medium. Here we present results studying actin network, a semiflexible polymer. We use a multiparticle - tracking approach to measure the local heterogeneities of the actin network. In another attempt to understand the heterogeneity of the network, the recently developed technique of two-point microrheology is used. Comparisons between the three different interpretation of the microrheological measurements are made and their further implications will be discussed.

[A18.002] MECHANICAL PROPERTIES OF ENTANGLED AND CROSSLINKED ACTIN SOLUTIONS

Applying dynamic light scattering and bulk rheology, we have investigated mechanical properties of semi-dilute actin solutions with and without the influence of a crosslinking protein copolymerized with actin in different concentrations. With the light scattering data, we have performed a linear mode analysis applying the theory for semiflexible polymer chains derived by Kroyamp;Frey. We have obtained estimates for various quantities like persistence and entanglement lengths that express mechanical properties of entangled actin gels. Theoretical considerations within the frame of the same model help us to explain the observed changes in mechanical properties upon addition of Cortexillin I, an actin-bundling protein from Dictyostelium. Adding this protein to actin in different concentrations, we have investigated the changes in mechanical properties, performing dynamic light scattering measurements at various wave- vectors.

[A18.003] Effect of Hydrophobic Modification Methods on the Gelation and Agregation of O-Methyl Cellulose

Gelation of o-metylcellulose (MC) in aqueous solution is believed to be caused primarily by the hydrophobic interaction between molecules containing methoxyl substitution. Recently it has been shown that different aggregates such as micelles, vesicles, and 'pearl-necklaces' are formed during different stages of the gelation process. In this paper we examine the effect of distribution on the gelation and aggregation processes.

Two types of molecules have been used: the first with block-like amphiphilic structure and the second with a more homogeneous distribution of substituted hydrophobic group. The systems were examined by means of Small Angle X-ray Scattering, Speckle pattern analysis and rheology. Large differences exist between the two types of systems indicating that gelation is more readily accomplished in the case of highly heterogeneous substituent distribution. It was also found that relaxation processes are very slow in these systems and structure evolves over periods extending to several days.

[A18.004] Self-assembled structures of cell cytoskeletal actin filaments by actin-crosslinking proteins

The actin cytoskeleton plays a major role in cell motility, cytokinesis and maintaining cell shape. These biological functions result from the interaction of actin with crosslinking proteins. We will focus on the structures formed in cell-free systems by the self-assembly of actin filaments (F-actin) in the presence of one such protein, alpha-actinin. We will also draw comparisons with the structures formed by the interaction of F-actin with model oligo-lysin polypeptides. At high alpha-actinin concentrations, confocal microscopy images show a highly interconnected network of F-actin bundles. Within the bundle, synchrotron small angle X-ray diffraction reveals a well-defined F-actin spacing of 30 nm. Surprisingly, this supramolecular assembly shows an unusually large intrinsic disorder that contrasts with the highly ordered lattices induced by oligo-lysin. We will discuss the implication of this difference for the formation of higher order mesostructures. Work supported by NIH GM59288, NSF DMR-9972246 and UC Biotechnology Grant 99-14.

[A18.005] Self-assembly of designed block polypeptides; control of final morphology with polypeptide secondary structure and molecular architecture

We are currently investigating different structural motifs (helix, sheet, and coil) and molecular architectures (diblock vs. triblock) available in synthetic block polypeptides as controllable parameters to define molecular self-assembly. Single molecule phase transitions between the different secondary structures add the intriguing potential of assembled structures that are responsive or “smart” to changes in their environment. Additional possibilities of biocompatibility and specific biological function are also available when designing macroscopic structures from the molecular, polypeptide level. The feasibility of dilute solution particle (micelles, vesicles) and concentrated mesoscale structure construction through molecular peptide design and assembly will be addressed. Small angle neutron and x-ray scattering results complemented with cryo transmission electron microscopy observations will be presented that focus on differences in assembly between alpha-helical, beta sheet formers, and random coil polypeptides both in diblock and triblock architectures.

[A18.006] Preparation, Stability, and Bio-Compatability of Block Copolymer Vesicles

Vesicles made completely from diblock copolymers – polymersomes – can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many–micron giants as well as monodisperse, ~100 nm vesicles of PEO-PEE (polyethyleneoxide – polyethylethylene) or PEO–PBD (polyethyleneoxide – polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can, but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a sub-population even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes, and several injections of 10 mg doses into rats show no ill-effect. The results are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG–driven micellization.

[A18.007] DNA Microarrays: Kinetics of Hybridization to a Polyelectyrolyte Brush

Gene sized DNA microarrays are charged biopolymer brushes whose hybridization to solution DNA is exploited in DNA chip technology of growing importance in genomics and medical applications such as drug development. The recognition of DNA sequences in solution requires the solution bound DNA to enter the brush and hybridize to a complementary sequence in the surface-tethered probe DNA. We present here a theoretical picture of these processes. Our interest is the case of oligonucleotide targets. Their entry into the brush is opposed by the loss of counterion entropy under the constraint of local electrical neutrality in the brush. The hybridization rate depends on the penetration depth of target DNA into the brush, which is governed by concentration of added salt, grafting density and relative size of probe and target. There are two classes of hybridization kinetics, either diffusion-controlled or dominated by the equilibrium profile, depending on conditions. We discuss optimal conditions to maximize hybridization rates and discrimination between perfect and slightly imperfect targets.

[A18.008] DNA Electrophoresis in Agarose Gels: Mobility vs. Length Dependence

Over the years, many different models have been applied to the migration of DNA fragments during gel electrophoresis. These models have been limited to describing DNA motion over specific size ranges. We propose a frictional and charge based model relating the electrophoretic mobility to length that fits data for DNA fragment lengths from 100 base pairs (bp) to 50 kilobase pairs (kbp). Excellent fits have been obtained from both published sources and experiments we have performed, with agarose gel concentrations of 0.5% to 1.5%. The length range of DNA for which the model works spans the range where a DNA fragment behaves like a semi-rigid rod to best described as a random coil polymer

[A18.009] DNA translocation across protein channels: How does a polymer worm through a hole?

Free energy barriers control the translocation of polymers through narrow channels. Based on an analogy with the classical nucleation and growth process, we have calculated the translocation time and its dependencies on the length, stiffness, and sequence of the polymer, solution conditions, and the strength of the driving electrochemical potential gradient. Our predictions will be compared with experimental results and prospects of reading polymer sequences.

[A18.010] Unbinding of Semiflexible Polymers

The unbinding transition of two semiflexible polymers is studied. The approach applies to biopolymers with large persistence lengths such as actin filaments or microtubuli. Using a mapping to the Klein-Kramers equation for Brownian motion the partition function can be calculated exactly in (1+1) dimensions. The critical exponents characterizing the unbinding from a potential well in the presence and absence of a hard wall can be infered for all spatial dimensions. The predicted criticality is confirmed numerically by transfer matrix calculations.

[A18.011] Designing toy proteins with several distinct stable states

Protein folding is traditionally associated with self-organization of a unique native state of a heteropolymer molecule. Apart from that, real proteins must also have the ability to change their conformations in response to certain functional stimuli. In order to capture this effect in the framework of a minimalistic lattice model we modified the sequence design method in such a way as to allow to encode more than one stable conformation in the sequence. The dynamics of polymer designed in this fashion is intimately related to the topology of its space of conformations. We examine relation between broken ergodicity of conformational movements upon volume restrictions and the kinetics of convertion between different imprinted compact states.

[A18.012] DNA Fluorescence Decay: Solution Versus Confined States

The time-resolved fluorescence anisotropy of DNA oligomers was studied using single photon counting methods after two-photon excitation of fluorescent-labelled DNA by a Ti:Sapphire femtosecond laser. The environment of the DNA (negatively-charged) was varied from bulk solution, to confinement within polyelectrolyte multilayers prepared by layer-by-layer assembly of polyanions and polycations. Key variables include temperature, ionic strength, and the degree of confinement as reflected in the thickness of the polyelectrolyte multilayer. Preliminary experiments are also reported using this technique to probe the hybridization of surface-attached DNA.

[A18.013] Anomalous x-ray scattering study on counterion condensed DNA

Because they are negatively charged, DNA molecules repel each other in monovalent salt solutions. However, in multivalent salt solutions [e.g. Mn, Co], DNA molecules attract one another and subsequently condense into liquid crystalline arrays. Nature makes use of these attractive interactions when packing DNA into confined spaces, such as those found in sperm heads and virus capsids. Established models of the electrostatic double layer, such as the Poisson-Boltzmann mean-field approach, fail to predict attraction between identically charged surfaces. Current speculation attributes such attraction to density fluctuations of the ionic atmospheres surrounding the DNA molecules, which give rise to a van der Waals-type interaction, or to the attraction brought about by a Wigner crystal-like arrangement of counterions on adjacent DNA strands. Therefore, an understanding of the spatial arrangement of counterions around DNA molecules in multivalent salt solutions is necessary to evaluate such hypotheses. We used anomalous x-ray scattering at the Advanced Photon Source (Argonne National Labs, IL), to measure of the counterion distribution around DNA molecules. These distributions should change as environmental conditions are changed from condensing to non-condensing, according to the aforementioned hypotheses. Preliminary analysis of our data suggests that the attraction can be attributed to a dispersion force induced by charge fluctuations of a diffuse ion cloud since long-range ordering of Co along the DNA chains could not be observed.

[A18.014] Nanomechanics of Cartilage : Investigation of Biomacromolecular Intermolecular Interactions via High-Resolution Force Spectroscopy

It is thought that the negatively charged, disaccharide macromolecules, glycosaminoglycans (GAGs), are the major determinant of the outstanding biomechanical properties of cartilage; i.e. its ability to resist compressive loading in vivo, responsible for 50-75 percent of the equilibrium compressive elastic modulus, as well as directly related to disease (i.e. osteoarthritis). The underlying molecular mechanism responsible for the outstanding compressive properties of cartilage arises from the unique nanomechanical properties of the constituent GAGs. In particular, intermolecular electrostatic repulsion due to the counterion electrical double layer of the charged polymers, as well as steric repulsion originating from configurational entropy. The technique of high-resolution force spectroscopy was employed to directly quanitfy these intermolecular interactions between biomimetic model end-grafted GAG "brush" surfaces with packing density 6nmx6nm-area per chain and probe tips of well-defined surface chemistry (e.g. sulfate-, methyl-, and GAG-functionalized).The effect of pH, ionic strength, grafting and charge density has been investigated.

[A18.015] Band structure effects on electron transport in DNA

Carrier transport in DNA is a subject of great importance, in part because of the crucial role that transport might play in the repair mechanism of radiation-induced damage. Recent experiments have reported results suggesting conflicting conclusions that DNA can act as either as an efficient conductor or a large bandgap semiconductor, making the question of whether DNA is metallic or semiconducting a contentious issue. We have carried out first-principles local-density functional simulations of infinitely long poly(G-C) DNA chains both in a pristine neutral state and in the presence of electron donors. Within the context of these simulations we discuss the effect that electron transfer might have on the electron transport properties of DNA molecules.

This work was supported by the US Office of Naval Research.

Part A of program listing