V. A. Raghunathan

Spontaneously Formed Monodisperse Biomimetic Unilamellar Vesicles: The Effect of Charge, Dilution, and Time

Using small-angle neutron scattering and dynamic light scattering, we have constructed partial structural phase diagrams of lipid mixtures composed of the phosphatidylcholines dimyristoyl and dihexanoyl doped with calcium ions (Ca2+) and/or the negatively charged lipid, dimyristoyl phosphatidylglycerol (DMPG). For dilute solutions (lipid concentration < or =1 wt %), spontaneously forming unilamellar vesicles (ULVs) were found, and their polydispersity was determined to be approximately 20%. The stability of the Ca2+- or DMPG-doped ULVs was monitored over a period of 4 days and their structural parameters (e.g., average outer radius, ) were found to be insensitive to the lipid concentration (Clp). However, doping the dimyristoyl/dihexanoyl system with both Ca2+ and DMPG resulted in ULVs whose was found to be Clp dependent. The of DMPG-doped ULVs remained unchanged over an extended period of time (at least 4 days), a good indication of their stability.

Temperature Driven Annealing of Perforations in Bicellar Model Membranes

Bicellar model membranes composed of 1,2-dimyristoylphosphatidylcholine (DMPC) and 1,2-dihexanoylphosphatidylcholine (DHPC), with a DMPC/DHPC molar ratio of 5, and doped with the negatively charged lipid 1,2-dimyristoylphosphatidylglycerol (DMPG), at DMPG/DMPC molar ratios of 0.02 or 0.1, were examined using small angle neutron scattering (SANS), (31)P NMR, and (1)H pulsed field gradient (PFG) diffusion NMR with the goal of understanding temperature effects on the DHPC-dependent perforations in these self-assembled membrane mimetics. Over the temperature range studied via SANS (300-330 K), these bicellar lipid mixtures exhibited a well-ordered lamellar phase. The interlamellar spacing d increased with increasing temperature, in direct contrast to the decrease in d observed upon increasing temperature with otherwise identical lipid mixtures lacking DHPC. (31)P NMR measurements on magnetically aligned bicellar mixtures of identical composition indicated a progressive migration of DHPC from regions of high curvature into planar regions with increasing temperature, and in accord with the mixed bicelle model (Triba, M. N.; Warschawski, D. E.; Devaux, P. E. Biophys. J.2005, 88, 1887-1901). Parallel PFG diffusion NMR measurements of transbilayer water diffusion, where the observed diffusion is dependent on the fractional surface area of lamellar perforations, showed that transbilayer water diffusion decreased with increasing temperature. A model is proposed consistent with the SANS, (31)P NMR, and PFG diffusion NMR data, wherein increasing temperature drives the progressive migration of DHPC out of high-curvature regions, consequently decreasing the fractional volume of lamellar perforations, so that water occupying these perforations redistributes into the interlamellar volume, thereby increasing the interlamellar spacing.

Phase behaviour of dipalmitoyl phosphatidylcholine (DPPC)-cholesterol membranes

We have determined the phase behaviour of dipalmitoyl phosphatidylcholine (DPPC)-cholesterol mixtures from small angle x-ray diffraction studies of oriented multilayers. A cholesterol induced modulated phase, denoted as Pβ, is obtained at intermediate cholesterol concentrations, which is distinct from the ripple () phase found in earlier studies on similar systems. We also report some confocal fluorescence microscopy observations on giant unilamellar vesicles (GUVs) made from these mixtures.

Effect of Ceramide on Nonraft Proteins

The currently accepted model of biological membranes involves a heterogeneous, highly dynamic organization, where certain lipids and proteins associate to form cooperative platforms (“rafts”) for cellular signaling or transport processes. Ceramides, a lipid species occurring under conditions of cellular stress and apoptosis, are considered to stabilize these platforms, thus modulating cellular function. The present study focuses on a previously unrecognized effect of ceramide generation. In agreement with previous studies, we find that ceramide leads to a depletion of sphingomyelin from mixtures with palmitoyl oleoyl phosphatidylcholine bilayers, forming a ceramide–sphingomyelin-rich gel phase that coexists with a fluid phase rich in palmitoyl oleoyl phosphatidylcholine. Interestingly, however, this latter phase has an almost fourfold smaller bending rigidity compared to a sphingomyelin–palmitoyl oleoyl phosphatidylcholine mixture lacking ceramide. The significant change of membrane bulk properties can have severe consequences for conformational equilibria of membrane proteins. We discuss these effects in terms of the lateral pressure profile concept for a simple geometric model of an ion channel and find a significant inhibition of its activity.

Novel structural features of the ripple phase of phospholipids

We have calculated, from X-ray diffraction data, the electron density maps of the ripple phase of dimyristoylphosphatidylcholine (DMPC) and palmitoyl-oleoyl phosphatidylcholine (POPC) multibilayers at different temperatures and fixed relative humidity. Our ana lysis of the maps establishes the existence of an average tilt of the hydrocarbon chains of the lipid molecules along the direction of the ripple wave vector, which we believe is responsible for the occurrence of asymmetric ripples in these systems.

Re-entrant Phase Behavior of a Concentrated Anionic Surfactant System with Strongly Binding Counterions†

The phase behavior of the anionic surfactant sodium dodecyl sulfate (SDS) in the presence of the strongly binding counterion p-toluidine hydrochloride (PTHC) has been examined using small-angle X-ray diffraction and polarizing microscopy. A hexagonal-to-lamellar transition on varying the PTHC to SDS molar ratio (alpha) occurs through a nematic phase of rodlike micelles (Nc) --> isotropic (I) --> nematic of disklike micelles (N(D)) at a fixed surfactant concentration (phi). The lamellar phase is found to coexist with an isotropic phase (I) over a large region of the phase diagram. Deuterium nuclear magnetic resonance investigations of the phase behavior at phi = 0.4 confirm the transition from N(C) to N(D) on varying alpha. The viscoelastic and flow behaviors of the different phases were examined. A decrease in the steady shear viscosity across the different phases with increasing alpha suggests a decrease in the aspect ratio of the micellar aggregates. From the transient shear stress response of the N() and N(D) nematic phases in step shear experiments, they were characterized to be tumbling and flow aligning, respectively. Our studies reveal that by tuning the morphology of the surfactant micelles strongly binding counterions modify the phase behavior and rheological properties of concentrated surfactant solutions.

Aligned Lipid—Water Systems

Under physiologically relevant conditions, membrane lipids normally form self-assembled bilayers where the lipids are usually in the disordered liquid crystalline or L α n this phase the lipid molecules are undergoing rapid translational diffusion and their fatty acid chains experience trans-gauche isomerizations [1–3]. Furthermore, ‘physiological relevance’ also implies that the lipid bilayers are ‘fully hydrated’ i.e. in excess water conditions. Although the condition of full hydration is easily met in liposomal preparations, by immersing the lipids in bulk water, this has not been the case for lipid bilayers aligned on solid supports and hydrated from water vapour [4,5]. The result of this was aligned bilayers exhibiting repeat spacings (d-spacings) which were less than their liposomal counterparts.

Reversible shear-induced crystallization above equilibrium freezing temperature in a lyotropic surfactant system

We demonstrate a unique shear-induced crystallization phenomenon above the equilibrium freezing temperature in weakly swollen isotropic and lamellar mesophases with bilayers formed in a cationic-anionic mixed surfactant system. Synchrotron rheological X-ray diffraction study reveals the crystallization transition to be reversible under shear (i.e., on stopping the shear, the nonequilibrium crystalline phase melts back to the equilibrium mesophase). This is different from the shear-driven crystallization below , which is irreversible. Rheological optical observations show that the growth of the crystalline phase occurs through a preordering of the phase to an phase induced by shear flow, before the nucleation of the phase. Shear diagram of the phase constructed in the parameter space of shear rate vs. temperature exhibits and transitions above the equilibrium crystallization temperature , in addition to the irreversible shear-driven nucleation of in the phase below . In addition to revealing a unique class of nonequilibrium phase transition, the present study urges a unique approach toward understanding shear-induced phenomena in concentrated mesophases of mixed amphiphilic systems.

Structure of mesh phases in cationic surfactant systems with strongly bound counterions: influence of the surfactant headgroup and the counterion.

We have studied the phase behavior of concentrated aqueous solutions of cetylpyridinium bromide (CPB) and sodium 3-hydroxy-2-naphthoate (SHN) using X-ray diffraction and polarizing optical microscopy. The phase behavior of this system is found to be very similar to that of the cetyltrimethylammonium bromide (CTAB)-SHN-water system, reported by us recently (Ghosh, S. K., et al. Langmuir, 2007, 23, 3606), but with the important difference that the mesh-like aggregates in the present system have square symmetry, instead of the hexagonal symmetry seen in the earlier case. A random mesh phase without long-range correlations of the in-plane structure, as well as an ordered mesh phase, where the mesh-like aggregates lock into a three-dimensional lattice, are observed, as in the CTAB-SHN-water system. The mesh-like aggregates do not form when the hydroxynaphthoate counterion is replaced by either salicylate or tosylate, which are also known to bind strongly to the surfactant micelle. Instead, the phase behavior of these ternary mixtures is akin to that of the CPB-water binary system; the only liquid crystalline phase observed being the hexagonal phase made up of cylindrical micelles. These results show the extreme sensitivity of the structure and stability of mesh phases to subtle changes in the interheadgroup interactions.

Chapter Seven - X-ray and Neutron Scattering Studies of Lipid–Sterol Model Membranes

Abstract Sterols are major components of many biomembranes and are known to play an important role in several biological processes. In order to understand the complex lipid–sterol interactions and their influence on membrane structure and properties, model membranes containing cholesterol and other sterols have been widely studied using a variety of experimental techniques. This chapter gives a brief review of X-ray and neutron scattering studies of these systems, highlighting the detailed structural information they provide.