Prabhanshu Shekhar

Structure and properties of tethered bilayer lipid membranes with unsaturated anchor molecules.

The self-assembled monolayers (SAMs) of new lipidic anchor molecule HC18 [Z-20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol] and mixed HC18/β-mercaptoethanol (βME) SAMs were studied by spectroscopic ellipsometry, contact angle measurements, reflection-absorption infrared spectroscopy, and electrochemical impedance spectroscopy (EIS) and were evaluated in tethered bilayer lipid membranes (tBLMs). Our data indicate that HC18, containing a double bond in the alkyl segments, forms highly disordered SAMs up to anchor/βME molar fraction ratios of 80/20 and result in tBLMs that exhibit higher lipid diffusion coefficients relative to those of previous anchor compounds with saturated alkyl chains, as determined by fluorescence correlation spectroscopy. EIS data shows the HC18 tBLMs, completed by rapid solvent exchange or vesicle fusion, form more easily than with saturated lipidic anchors, exhibit excellent electrical insulating properties indicating low defect densities, and readily incorporate the pore-forming toxin α-hemolysin. Neutron reflectivity measurements on HC18 tBLMs confirm the formation of complete tBLMs, even at low tether compositions and high ionic lipid compositions. Our data indicate that HC18 results in tBLMs with improved physical properties for the incorporation of integral membrane proteins (IMPs) and that 80% HC18 tBLMs appear to be optimal for practical applications such as biosensors where high electrical insulation and IMP/peptide reconstitution are imperative.

Continuous distribution model for the investigation of complex molecular architectures near interfaces with scattering techniques.

Biological membranes are composed of a thermally disordered lipid matrix and therefore require non-crystallographic scattering approaches for structural characterization with x-rays or neutrons. Here we develop a continuous distribution (CD) model to refine neutron or x-ray reflectivity data from complex architectures of organic molecules. The new model is a flexible implementation of the composition-space refinement of interfacial structures to constrain the resulting scattering length density profiles. We show this model increases the precision with which molecular components may be localized within a sample, with a minimal use of free model parameters. We validate the new model by parameterizing all-atom molecular dynamics (MD) simulations of bilayers and by evaluating the neutron reflectivity of a phospholipid bilayer physisorbed to a solid support. The determination of the structural arrangement of a sparsely-tethered bilayer lipid membrane (stBLM) comprised of a multi-component phospholipid bilayer anchored to a gold substrate by a thiolated oligo(ethylene oxide) linker is also demonstrated. From the model we extract the bilayer composition and density of tether points, information which was previously inaccessible for stBLM systems. The new modeling strategy has been implemented into the ga_refl reflectivity data evaluation suite, available through the National Institute of Standards and Technology (NIST) Center for Neutron Research (NCNR).

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζcyt) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζcyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζcyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζcyt to the bilayers in dynamic equilibrium: one in which ζcyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζcyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Structure of the PTEN Tumor Suppressor Associated with the Fluid Lipid Membranes

e tumor suppressor PTEN1 is a phosphatase involved in the regulation of PI(3,4,5)P3. It consists of a phosphatase and a C2 domain that interact synergistically with anionic lipids in membranes. A single-point mutation of the wild type (wt) protein, H93R, has the same secondary structure but significantly reduced enzyme activity. As for many membrane proteins, the crystal structure of (a truncated) PTEN has been determined2, but the association of the protein with lipid membranes has only been indirectly inferred. We study the association of wt PTEN and H93R with membranes using neutron reflectometry (NR) of tethered bilayer lipid membranes (tBLMs)3,4 which are long-term stable and retain their fluidity with in-plane dynamics similar to that in vesicles.5 Surface Plasmon Resonance (SPR) spectroscopy has also been used to investigate the binding of these two variants of PTEN.Data analysis uses a composition-space model that resolves the thermally disordered membrane with Angstrom resolution,6 enabling us to characterize PTEN association with the bilayer in its physiologically relevant, disordered state. The crystal structure serves as a starting point for model refinement. Computational techniques are used to explore the conformational flexibility of the peptide stretches deleted for crystallization. We observed slight differences in the peripheral association of H93R and wt PTEN with the bilayers headgroup region and speculate how these may be related to their functional distinctions.(1) I. Sansal et al., 2004, J. Clin. Oncology 22:2954.(2) J. Lee,et al., 1999, Cell 99:323.(3) D. J. McGillivray et al., 2007, Biointerphases 2:21.(4) F. Heinrich et al., 2009, Langmuir 25:4219.(5) S. Shenoy et al., 2010, Soft Matter 6, 1263.(6) F. Heinrich et al., J. Appl. Phys., submitted.