Hongjun Liang

Like-charge attraction between polyelectrolytes induced by counterion charge density waves

Electrostatics in aqueous media is commonly understood in terms of screened Coulomb interactions, where like-charged objects, such as polyelectrolytes, always repel. These intuitive expectations are based on mean field theories, such as the Poisson–Boltzmann formalism, which are routinely used in colloid science and computational biology [Israelachvili, J. (1992) Intermolecular and Surface Forces (Academic, London), 2nd ed.]. Like-charge attractions, however, have been observed in a variety of systems [Gelbart, W. M., Bruinsma, R. F., Pincus, P. A. & Parsegian, V. A. (2000) Phys. Today 53, 38–44]. Intense theoretical scrutiny over the last 30 years suggests that counterions play a central role, but no consensus exists for the precise mechanism. We have directly observed the organization of multivalent ions on cytoskeletal filamentous actin (a well defined biological polyelectrolyte) by using synchrotron x-ray diffraction and discovered an unanticipated symmetry-breaking collective counterion mechanism for generating attractions. Surprisingly, the counterions do not form a lattice that simply follows actins helical symmetry; rather, the counterions organize into “frozen” ripples parallel to the actin filaments and form 1D charge density waves. Moreover, this 1D counterion charge density wave couples to twist distortions of the oppositely charged actin filaments. This general cooperative molecular mechanism is analogous to the formation of polarons in ionic solids and mediates attractions by facilitating a “zipper-like” charge alignment between the counterions and the polyelectrolyte charge distribution. We believe these results can fundamentally impinge on our general understanding of electrostatics in aqueous media and are relevant to a wide range of colloidal and biomedical processes.

Enhanced Environmental Mobility of Carbon Nanotubes in the Presence of Humic Acid and Their Removal from Aqueous Solution

Carbon nanotubes (CNTs) are important structural blocks for the preparation of composites with unique optical, electrical, and mechanical properties, and their production is expected to increase drastically in the years to come. [1] This may increase the risk of human and environmental exposure to CNTs. [1d–h] CNTs are extremely hydrophobic and prone to aggregation, as they are subject to higher van der Waals forces along their length axis, and therefore are not readily dispersed in aqueous or non-aqueous solutions; this has been a major obstacle for the application of CNTs in industry. [2] As a result, significant attention has been directed towards methods of CNT dispersion in aqueous solution. Two methods of exohedral functionalization of CNTs have been developed to disperse them; covalent [3] and non-covalent methods. [4] Non-covalent methods are more desirable since they incur little damage to the CNTs’ intrinsic structures and properties. Dispersants tested in the laboratory for non-covalent functionalization of CNTs include surfactants, synthetic polymers, and biopolymers. [4] Therefore, even though some studies have shown that CNTs are biologically active and cause toxic responses in some cell cultures, [5] CNTs are not usually considered as potential environmental toxins in the aqueous and soil environment [6]

The directed cooperative assembly of proteorhodopsin into 2D and 3D polarized arrays

Proteorhodopsin is the membrane protein used by marine bacterioplankton as a light-driven proton pump. Here, we describe a rapid cooperative assembly process directed by universal electrostatic interactions that spontaneously organizes proteorhodopsin molecules into ordered arrays with well defined orientation and packing density. We demonstrate the charge density-matching mechanism that selectively controls the assembly process. The interactions among different components in the system are tuned by varying their charge densities to yield different organized transmembrane protein arrays: (i) a bacteriorhodopsin purple membrane-like structure where proteorhodopsin molecules are cooperatively arranged with charged lipids into a 2D hexagonal lattice; (ii) selected liquid-crystalline states in which crystalline lamellae made up of the coassembled proteorhodopsin and charged lipid molecules are coupled three-dimensionally with polarized proteorhodopsin orientation persisting through the macroscopic scale. Understanding this rapid electrostatically driven assembly process sheds light on organizing membrane proteins in general, which is a prerequisite for membrane protein structural and mechanistic studies as well as in vitro applications.

Thrombin generation and fibrin formation under flow on biomimetic tissue factor-rich surfaces.

Blood flow regulates coagulation and fibrin assembly by controlling the rate of transport of zymogens, enzymes and plasma proteins to and from the site of an injury.

Self-directed reconstitution of proteorhodopsin with amphiphilic block copolymers induces the formation of hierarchically ordered proteopolymer membrane arrays.

Manipulating recognition and transport at the nanoscale holds great promise for technological breakthroughs in energy conversion, catalysis, and information processing. Living systems evolve specialized membrane proteins (MPs) embedded in lipid bilayers to exquisitely control communications across the insulating membrane boundaries. Harnessing MP functions directly in synthetic systems opens up enormous opportunities for nanotechnology, but there exist fundamental challenges of how to address the labile nature of lipid bilayers that renders them of inadequate value under a broad range of harsh non-biological conditions, and how to reconstitute MPs coherently in two or three dimensions into non-lipid-based artificial membranes. Here we show that amphiphilic block copolymers can be designed to direct proteorhodopsin reconstitution and formation of hierarchically ordered proteopolymer membrane arrays spontaneously, even when the membrane-forming polymer blocks are in entangled states. These findings unfold a viable approach for the development of robust and chemically versatile nanomembranes with MP-regulated recognition and transport performance.

Structure and dynamics of condensed multivalent ions within polyelectrolyte bundles: a combined x-ray diffraction and solid-state NMR study

Like-charged polyelectrolytes can attract and condense into compact ordered states via counterion-mediated interactions (Gelbart et al 2000 Phys. Today 53 38–44). Recent examples include DNA toroids and F-actin bundles. We have investigated the structure and dynamics of condensed divalent ions within F-actin polyelectrolyte bundles. Using synchrotron x-ray diffraction, the structural organization of Ba 2+ ions on F-actin has been directly observed. The Ba 2+ ions organize into counterion charge density waves (CDWs) parallel to the actin filaments. Moreover, these 1D counterion charge density waves couple to twist deformations of the oppositely charged actin filaments, and mediate attractions by effecting a ‘zipper-like’ charge alignment between the counterions and the polyelectrolyte charge distribution. We have also examined condensed divalent 25 Mg ions within F-actin bundles using solid-state NMR. Preliminary measurements indicate tha tt he longitudinal relaxation time T1 of Mg 2+ ions decreases by approximately an order of magnitude as they organize into th eC DW state within condensed F-actin bundles. The measured value of T1 for Mg 2+ ions in the CDW is intermediate between typical liquid-like and solid-like values. (Some figures in this article are in colour only in the electronic version)

Synthesis of cationic poly(4-vinylpyridine)-functionalized colloidal particles by emulsion polymerization with reactive block copolymer for protein adsorption.

We report here a novel protein sequestration method using polymeric colloidal particles prepared by emulsion polymerization with reactive block copolymers. Specifically, poly(4-vinyl-N-ethylpyridine bromide)-block-polymethylacrylate block copolymers were synthesized from reversible addition-fragmentation chain transfer polymerization and used as emulsifiers for emulsion polymerization of poly(4-vinyl-N-ethylpyridine bromide)-functionalized polymeric colloidal particles. The particles have high and stable zeta potentials when dispersed in solution, regardless of pH variations. As a result, the polymeric colloids demonstrate a high affinity for oppositely charged proteins, even though the isoelectric points of proteins may vary greatly. We show here that BSA can be sequestered highly efficiently with a maximum binding capacity (~900 mg/g). The adsorbed protein is easily released, and the polymeric colloids are regenerated after washing with a buffer solution of high ionic strength. These properties may prompt this type of novel macromolecule-functionalized colloids to be utilized for effective protein adsorption and separation.

Interface for Light-Driven Electron Transfer by Photosynthetic Complexes Across Block Copolymer Membranes

Incorporation of membrane proteins into nanodevices to mediate recognition and transport in a collective and scalable fashion remains a challenging problem. We demonstrate how nanoscale photovoltaics could be designed using robust synthetic nanomembranes with incorporated photosynthetic reaction centers (RCs). Specifically, RCs from Rhodobacter sphaeroides are reconstituted spontaneously into rationally designed polybutadiene membranes to form hierarchically organized proteopolymer membrane arrays via a charge-interaction-directed reconstitution mechanism. Once incorporated, the RCs are fully active for prolonged periods based upon a variety of spectroscopic measurements, underscoring preservation of their 3D pigment configuration critical for light-driven charge transfer. This result provides a strategy to construct solar conversion devices using structurally versatile proteopolymer membranes with integrated RC functions to harvest broad regions of the solar spectrum.

An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein

To further improve the selectivity and throughput of phosphopeptide analysis for the samples from real-time cell lysates, here we demonstrate a highly efficient method for phosphopeptide enrichment via newly synthesized magnetite microparticles and the concurrent mass spectrometric analysis. The magnetite microparticles show excellent magnetic responsivity and redispersibility for a quick enrichment of those phosphopeptides in solution. The selectivity and sensitivity of magnetite microparticles in phosphopeptide enrichment are first evaluated by a known mixture containing both phosphorylated and nonphosphorylated proteins. Compared with the titanium dioxide-coated magnetic beads commercially available, our magnetite microparticles show a better specificity toward phosphopeptides. The selectively-enriched phosphopeptides from tryptic digests of β-casein can be detected down to 0.4 fmol μl⁻¹, whereas the recovery efficiency is approximately 90% for monophosphopeptides. This magnetite microparticle-based affinity technology with optimized enrichment conditions is then immediately applied to identify all possible phosphorylation sites on a signal protein isolated in real time from a stress-stimulated mammalian cell culture. A large fraction of peptides eluted from the magnetic particle enrichment step were identified and characterized as either single- or multiphosphorylated species by tandem mass spectrometry. With their high efficiency and utility for phosphopeptide enrichment, the magnetite microparticles hold great potential in the phosphoproteomic studies on real-time samples from cell lysates.

Structure and Dynamics of Extracellular Loops in Human Aquaporin-1 from Solid-State NMR and Molecular Dynamics

Multiple moderate-resolution crystal structures of human aquaporin-1 have provided a foundation for understanding the molecular mechanism of selective water translocation in human cells. To gain insight into the interfacial structure and dynamics of human aquaporin-1 in a lipid environment, we performed nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Using magic angle spinning solid-state NMR, we report a near complete resonance assignment of the human aquaporin-1. Chemical shift analysis of the secondary structure identified pronounced deviations from crystallographic structures in extracellular loops A and C, including the cis Y37-P38 bond in loop A, as well as ordering and immobilization of loop C. Site-specific H/D exchange measurements identify a number of protected nitrogen-bearing side chains and backbone amide groups, involved in stabilizing the loops. A combination of molecular dynamics simulations with NMR-derived restraints and filtering based on solvent accessibility allowed for the determination of a structural model of extracellular loops largely consistent with NMR results. The simulations reveal loop stabilizing interactions that alter the extracellular surface of human AQP1, with possible implications for water transport regulation through the channel. Modulation of water permeation may occur as a result of rearrangement of side chains from loop C in the extracellular vestibule of hAQP1, affecting the aromatic arginine selectivity filter.