Erik B. Watkins

Membrane texture induced by specific protein binding and receptor clustering: active roles for lipids in cellular function

Biological membranes are complex, self-organized structures that define boundaries and compartmentalize space in living matter. Composed of a wide variety of lipid and protein molecules, these responsive surfaces mediate transmembrane signaling and material transport within the cell and with its environment. It is well known that lipid membrane properties change as a function of composition and phase state, and that protein-lipid interactions can induce changes in the membrane’s properties and biochemical response. Here, molecular level changes in lipid organization induced by multivalent toxin binding were investigated using grazing incidence X-ray diffraction. Structural changes to lipid monolayers at the air-water interface and bilayers at the solid-water interface were studied before and after specific binding of cholera toxin to membrane embedded receptors. At biologically relevant surface pressures, protein binding perturbed lipid packing within monolayers and bilayers resulting in topological defects and the emergence of a new orientationally textured lipid phase. In bilayers this altered lipid order was transmitted from the receptor laden exterior membrane leaflet to the inner leaflet, representing a potential mechanism for lipid mediated outside-in signaling by multivalent protein binding. It is further hypothesized that cell-surface micro-domains exhibiting this type of lipid order may serve as nucleation sites for vesicle formation in clathrin independent endocytosis of cholera toxin.

Structure and Thermodynamics of Lipid Bilayers on Polyethylene Glycol Cushions: Fact and Fiction of PEG Cushioned Membranes

In developing well hydrated polymer cushioned membranes, structural studies are often neglected. In this work, neutron and X-ray reflectivity studies reveal that hybrid bilayer/polyethylene glycol (PEG) systems created from mixtures of phospholipids and PEG conjugated lipopolymers do not yield a hydrated cushion beneath the bilayer unless the terminal ends of the lipopolymers are functionalized with reactive end groups and can covalently bind (tether) to the underlying support surface. While reactive PEG tethered systems yielded bilayers with near complete surface coverage, a bimodal distribution of heights with sub-micrometer lateral dimensions was observed consisting of cushioned membrane domains and uncushioned regions in close proximity to the support. The membrane fraction cushioned by the hydrated polymer could be controlled by adjusting the molar ratio of lipopolymer in the bilayer. A general phase diagram based on the free energy of the various configurations is derived that qualitatively predicts the observed behavior and the resulting structure of such systems a priori. As further evidenced by ellipsometry, atomic force and fluorescence microscopy, the tethered system provides a simple means for fabricating small cushioned domains within a membrane.

Structure and stability of phospholipid bilayers hydrated by a room-temperature ionic liquid/water solution: a neutron reflectometry study.

Neutron reflectometry (NR) measurements were carried out to probe the structure and stability of two model biomembranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) phospholipid bilayers hydrated by water solutions of two prototypical room-temperature ionic liquids (RTILs), namely, 1-butyl-3-methyl-imidazolium chloride ([bmim][Cl]) and choline chloride ([Chol][Cl]) at concentrations of 0.1 M and 0.5 M, respectively. The raw data were analyzed by fitting a distribution of scattering length densities arising from the different chemical species in the system. The results of this analysis show that (a) for all systems and concentrations that we considered, the thickness of the bilayers shrinks by ∼1 Å upon dissolving the ionic liquid into water and that (b) the RTIL ions enter the bilayer, finding their way to a preferred location in the lipid range that is nearly independent of the lipid and of the [bimim](+) or [Chol](+) choice. The volume fraction of RTIL sorbed in/on the bilayer, however, does depend on the lipid, but, again, is the same for [bmim][Cl] and for [Chol][Cl]. Thus, the RTIL occupies ∼5% of the bilayer volume in POPC, rising to ∼10% in DMPC. Repeating the measurements and data analysis after rinsing in pure water shows that the changes in the bilayer due to the RTIL sorption are irreversible and that a measurable amount of IL remains in the lipid fraction, that is, ∼2.5% of the bilayer volume in POPC and ∼8% in DMPC.

pH Responsive Polymer Cushions for Probing Membrane Environment Interactions

A robust and straightforward method for the preparation of lipid membranes upon dynamically responsive polymer cushions is reported. Structural characterization demonstrates that complete, well-packed membranes with tunable mobility can be constructed on the polymeric cushion. With this system, membrane conformational changes induced by cellular cytoskeleton interactions can be modeled. The membrane can be tailored to screen the cushion from changes in pH or allow rapid response to the pH environment by incorporation of protein ion channels. This elementary system offers a means to replicate the conformational changes that occur with the cellular cytoskeleton and has great potential for fundamental biophysical studies of membrane properties and membrane-protein interactions decoupled from the underlying solid support.

Part I: An X-Ray Scattering Study of Cholera Toxin Penetration and Induced Phase Transformations in Lipid Membranes

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM(1). However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM(1) at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B(5) pentamer, was also observed.

Carbohydrate Conformation and Lipid Condensation in Monolayers Containing Glycosphingolipid Gb3: Influence of Acyl Chain Structure

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3s influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3s capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environments impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.

Time-resolved specular and off-specular neutron reflectivity measurements on deuterated polystyrene and poly(vinyl methyl ether) blend thin films during dewetting process

We performed time-resolved specular and off-specular neutron reflectivity measurements on blend thin films 42 and 98 nm thick of deuterated polystyrene and poly(vinyl methyl ether) during dewetting process induced by the phase separation in two phase region using a time-of-flight neutron reflectometer. In the specular measurements we found that the phase separation directed to the depth direction occurred near the air interface as well as near the Si substrate during the incubation period before dewetting. In addition we also found that the phase separation occurred asymmetrically at the two interfaces and inhomogeneously in the film plane, showing that the dewetting was induced by the composition fluctuation mechanism. Off-specular reflectivity was analyzed, for the first time, to evaluate kinetics of structure formation in the film plane during the dewetting process. We found in the analysis that the droplets formation in micrometer scale occurred in the late stage of dewetting.

Equilibrium or Quenched: Fundamental Differences between Lipid Monolayers, Supported Bilayers, and Membranes

In this work, we establish fundamental differences between the structure and packing of lipids in monolayers, supported bilayers, and multilayer films. High resolution grazing incidence X-ray diffraction reveals that monolayer structure is largely retained upon deposition onto substrates with the area per molecule controlled by deposition pressure. Such structural changes are consistent with a quenched rather than equilibrated supported membrane structure. Supported bilayers formed by vesicle fusion exhibit structural similarity to bilayers deposited at 38 mN/m, whereas packing in lipid multilayers more closely resembled bilayers deposited below 30 mN/m. At the molecular level, coupling between opposing lipid acyl chains is observed for all deposition pressures with the outer leaflet templating on the inner leaflet. Leaflet coupling induces a small condensation in the area per lipid molecule and a surprising increase in acyl chain tilt. Moreover, supported lipid bilayers exhibit preferential acyl chain alignment: the system cannot be modeled with freely rotating acyl chains as in free-standing lipid monolayers. Such acyl chain alignment is consistent with orientational texture of lipid tilt directors at larger length scales. These findings clearly demonstrate that supported, gel-phase bilayer membrane structure can be controlled and maintained by deposition onto solid supports and that increasing surface pressure induces preferential alignment of the acyl chains both within and between membrane leaflets.

Physical properties of archaeal tetraether lipid membranes as revealed by differential scanning and pressure perturbation calorimetry, molecular acoustics, and neutron reflectometry: effects of pressure and cell growth temperature.

The polar lipid fraction E (PLFE) is a major tetraether lipid component in the thermoacidophilic archaeon Sulfolobus acidocaldarius. Using differential scanning and pressure perturbation calorimetry as well as ultrasound velocity and density measurements, we have determined the compressibilities and volume fluctuations of PLFE liposomes derived from different cell growth temperatures (T(g) = 68, 76, and 81 °C). The compressibility and volume fluctuation values of PLFE liposomes, which are substantially less than those detected from diester lipid membranes (e.g., DPPC), exhibit small but significant differences with T(g). Among the three T(g)s employed, 76 °C leads to the least compressible and most tightly packed PLFE membranes. This temperature is within the range for optimal cell growth (75-80 °C). It is known that a decrease in T(g) decreases the number of cyclopentane rings in archael tetraether lipids. Thus, our data enable us to present the new view that membrane packing in PLFE liposomes varies with the number of cyclopentane rings in a nonlinear manner, reaching maximal tightness when the tetraether lipids are derived from cells grown at optimal T(g)s. In addition, we have studied the effects of pressure on total layer thickness, d, and neutron scattering length density, ρ(n), of a silicon-D(2)O interface that is covered with a PLFE membrane using neutron reflectometry (NR). At 55 °C, d and ρ(n) are found to be rather insensitive to pressure up to 1800 bar, suggesting minor changes of the thickness of the membranes hydrophobic core and headgroup orientation upon compression only.

Interactions of Small Dendrimers with Sodium Dodecyl Sulfate at the Air-Water Interface.

We have determined how the bulk behavior of mixtures of small cationic poly(amidoamine) dendrimers (generation 2, PAMAM-G2) and sodium dodecyl sulfate (SDS) affects the structure and composition of the adsorbed layers at the air-water interface. The aim is to reveal how the size of a well-defined hyperbranched polyelectrolyte affects the interfacial and bulk solution behavior of mixtures with oppositely charged surfactants, when the size of the polyelectrolyte approaches that of the surfactant. A combination of electrophoretic mobility, UV-vis spectroscopy, dynamic light scattering, and small-angle X-ray scattering measurements have been employed to characterize the interactions in the bulk solution. PAMAM-G2 associates strongly with SDS in the bulk, forming large aggregates where the size and the charge depend on the bulk composition. We show that kinetically trapped aggregates can be formed at compositions outside the equilibrium two-phase region, and the positively charged aggregates are larger than the negative ones. Surface tensiometry, neutron reflectometry, and ellipsometry have been used to reveal the properties of the interfacial layers. The interfacial structures formed depend strongly on the bulk composition: structured layers are present for samples inside the two-phase region, whereas intact nanostructured aggregates adsorb for samples just outside the two-phase region. The interfacial behavior of PAMAM-G2/SDS mixtures is compared with that of small amines or multivalent ions and oppositely charged surfactants. The implications of aggregate adsorption, dissociation, and spreading processes are discussed as well as the potential of small dendrimers for applications involving the delivery of functional molecules to interfaces.