Publications

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The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Here, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we found that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup-membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the Lo phase.

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (Lo)-liquid disordered (Ld) phase separated lipid bilayers when the following particles of increasing size bind to either the Lo or Ld phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu2+ chelating mechanism, of lipids that specifically partition into either the Lo phase or Ld phase. The degree of steric pressure was controlled by varying the size of the bound particle (10-240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed Lo phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.

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Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (> 4 μm) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (>4 μm) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (>100 μm).

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu2+, chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10-22 to 1.5 × 10-20 J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.

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Advances in technology for electrochemical energy storage require increased understanding of electrolyte/electrode interfaces, including the electric double layer structure, and processes involved in charging of the interface, and the incorporation of this understanding into quantitative models. Simplified models such as Helmholtz's electric double-layer (EDL) concept don't account for the molecular nature of ion distributions, solvents, and electrode surfaces and therefore cannot be used in predictive, high-fidelity simulations for device design. This report presents theoretical results from models that explicitly include the molecular nature of the electrical double layer and predict critical electrochemical quantities such as interfacial capacitance. It also describes development of experimental tools for probing molecular properties of electrochemical interfaces through optical spectroscopy. These optical experimental methods are designed to test our new theoretical models that provide descriptions of the electric double layer in unprecedented detail.

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The pulsed photolytic chlorine-initiated oxidation of methyl-tert-butyl ketone (MTbuK), di-tert-butyl ketone (DTbuK), and a series of partially deuterated diethyl ketones (DEK) is studied in the gas phase at 8 Torr and 550-650 K. Products are monitored as a function of reaction time, mass, and photoionization energy using multiplexed photoionization mass spectrometry with tunable synchrotron ionizing radiation. The results establish that the primary 3-oxoalkyl radicals of those ketones, formed by abstraction of a hydrogen atom from the carbon atom in γ-position relative to the carbonyl oxygen, undergo a rapid rearrangement resulting in an effective 1,2-acyl group migration, similar to that in a Dowd-Beckwith ring expansion. Without this rearrangement, peroxy radicals derived from MTbuK and DTbuK cannot undergo HO2 elimination to yield a closed-shell unsaturated hydrocarbon coproduct. However, not only are these coproducts observed, but they represent the dominant oxidation channels of these ketones under the conditions of this study. For MTbuK and DTbuK, the rearrangement yields a more stable tertiary radical, which provides the thermodynamic driving force for this reaction. Even in the absence of such a driving force in the oxidation of partially deuterated DEK, the 1,2-acyl group migration is observed. Quantum chemical (CBS-QB3) calculations show the barrier for gas-phase rearrangement to be on the order of 10 kcal mol-1. The MTbuK oxidation experiments also show several minor channels, including β-scission of the initial radicals and cyclic ether formation. © 2013 American Chemical Society.

The characterization of liposomes was undertaken using in-situ microfluidic transmission electron microscopy. Liposomes were imaged without contrast enhancement staining or cryogenic treatment, allowing for the observation of functional liposomes in an aqueous environment. The stability and quality of the liposome structures observed were found to be highly dependent on the surface and liposome chemistries within the liquid cell. The successful imaging of liposomes suggests the potential for the extension of in-situ microfluidic TEM to a wide variety of other biological and soft matter systems and processes. © 2013 American Chemical Society.

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Cell membranes are dynamic substrates that achieve a diverse array of functions through multi-scale reconfigurations. We explore the morphological changes that occur upon protein interaction to model membrane systems that induce deformation of their planar structure to yield nanotube assemblies. In the two examples shown in this report we will describe the use of membrane adhesion and particle trajectory to form lipid nanotubes via mechanical stretching, and protein adsorption onto domains and the induction of membrane curvature through steric pressure. Through this work the relationship between membrane bending rigidity, protein affinity, and line tension of phase separated structures were examined and their relationship in biological membranes explored.

The innate immune system represents our first line of defense against microbial pathogens, and in many cases is activated by recognition of pathogen cellular components (dsRNA, flagella, LPS, etc.) by cell surface membrane proteins known as toll-like receptors (TLRs). As the initial trigger for innate immune response activation, TLRs also represent a means by which we can effectively control or modulate inflammatory responses. This proposal focused on TLR4, which is the cell-surface receptor primarily responsible for initiating the innate immune response to lipopolysaccharide (LPS), a major component of the outer membrane envelope of gram-negative bacteria. The goal was to better understand TLR4 activation and associated membrane proximal events, in order to enhance the design of small molecule therapeutics to modulate immune activation. Our approach was to reconstitute the receptor in biomimetic systems in-vitro to allow study of the structure and dynamics with biophysical methods. Structural studies were initiated in the first year but were halted after the crystal structure of the dimerized receptor was published early in the second year of the program. Methods were developed to determine the association constant for oligomerization of the soluble receptor. LPS-induced oligomerization was observed to be a strong function of buffer conditions. In 20 mM Tris pH 8.0 with 200 mM NaCl, the onset of receptor oligomerization occurred at 0.2 uM TLR4/MD2 with E coli LPS Ra mutant in excess. However, in the presence of 0.5 uM CD14 and 0.5 uM LBP, the onset of receptor oligomerization was observed to be less than 10 nM TLR4/MD2. Several methods were pursued to study LPS-induced oligomerization of the membrane-bound receptor, including CryoEM, FRET, colocalization and codiffusion followed by TIRF, and fluorescence correlation spectroscopy. However, there approaches met with only limited success.

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A novel hyperspectral fluorescence microscope for high-resolution 3D optical sectioning of cells and other structures has been designed, constructed, and used to investigate a number of different problems. We have significantly extended new multivariate curve resolution (MCR) data analysis methods to deconvolve the hyperspectral image data and to rapidly extract quantitative 3D concentration distribution maps of all emitting species. The imaging system has many advantages over current confocal imaging systems including simultaneous monitoring of numerous highly overlapped fluorophores, immunity to autofluorescence or impurity fluorescence, enhanced sensitivity, and dramatically improved accuracy, reliability, and dynamic range. Efficient data compression in the spectral dimension has allowed personal computers to perform quantitative analysis of hyperspectral images of large size without loss of image quality. We have also developed and tested software to perform analysis of time resolved hyperspectral images using trilinear multivariate analysis methods. The new imaging system is an enabling technology for numerous applications including (1) 3D composition mapping analysis of multicomponent processes occurring during host-pathogen interactions, (2) monitoring microfluidic processes, (3) imaging of molecular motors and (4) understanding photosynthetic processes in wild type and mutant Synechocystis cyanobacteria.

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We have developed a new nanotagging technology for detecting and imaging the self-organization of proteins and other components of membranes at nanometer resolution for the purpose of investigating cell signaling and other membrane-mediated biological processes. We used protein-, lipid-, or drug-bound porphyrin photocatalysts to grow in-situ nanometer-sized metal particles, which reveal the location of the porphyrin-labeled molecules by electron microscopy. We initially used photocatalytic nanotagging to image assembled multi-component proteins and to monitor the distribution of lipids and porphyrin labels in liposomes. For example, by exchanging the heme molecules in hemoproteins with a photocatalytic tin porphyrin, a nanoparticle was grown at each heme site of the protein. The result obtained from electron microscopy for a tagged multi-subunit protein such as hemoglobin is a symmetric constellation of a specific number of nanoparticle tags, four in the case of the hemoglobin tetramer. Methods for covalently linking photocatalytic porphyrin labels to lipids and proteins were also developed to detect and image the self-organization of lipids, protein-protein supercomplexes, and membrane-protein complexes. Procedures for making photocatalytic porphyrin-drug, porphyrin-lipid, and porphyrin-protein hybrids for non-porphyrin-binding proteins and membrane components were pursued and the first porphyrin-labeled lipids was investigated in liposomal membrane models. Our photocatalytic nanotagging technique may ultimately allow membrane self-organization and cell signaling processes to be imaged in living cells. Fluorescence and plasmonic spectra of the tagged proteins might also provide additional information about protein association and membrane organization. In addition, a porphyrin-aspirin or other NSAID hybrid may be used to grow metal nanotags for the pharmacologically important COX enzymes in membranes so that the distribution of the protein can be imaged at the nanometer scale.

The formation and functions of living materials and organisms are fundamentally different from those of synthetic materials and devices. Synthetic materials tend to have static structures, and are not capable of adapting to the functional needs of changing environments. In contrast, living systems utilize energy to create, heal, reconfigure, and dismantle materials in a dynamic, non-equilibrium fashion. The overall goal of the project was to organize and reconfigure functional assemblies of nanoparticles using strategies that mimic those found in living systems. Active assembly of nanostructures was studied using active biomolecules to drive the organization and assembly of nanocomposite materials. In this system, kinesin motor proteins and microtubules were used to direct the transport and interactions of nanoparticles at synthetic interfaces. In addition, the kinesin/microtubule transport system was used to actively assemble nanocomposite materials capable of storing significant elastic energy. Novel biophysical measurement tools were also developed for measuring the collective force generated by kinesin motor proteins, which will provide insight on the mechanical constraints of active assembly processes. Responsive reconfiguration of nanostructures was studied in terms of using active biomolecules to mediate the optical properties of quantum dot (QD) arrays through modulation of inter-particle spacing and associated energy transfer interaction. Design rules for kinesin-based transport of a wide range of nanoscale cargo (e.g., nanocrystal quantum dots, micron-sized polymer spheres) were developed. Three-dimensional microtubule organizing centers were assembled in which the polar orientation of the microtubules was controlled by a multi-staged assembly process. Overall, a number of enabling technologies were developed over the course of this project, and will drive the exploitation of energy-driven processes to regulate the assembly, disassembly, and dynamic reorganization of nanomaterials.