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Even after decades of research, a comprehensive mechanism that elucidates the underpinnings of signaling through the cell membrane is still elusive. Here, we address a simple question- “how does the ectodomain of a membrane-associated protein consisting of multiple domains and connected by flexible linkers stand ‘upright’ on the membrane?”. Our analysis based on large amount of available functional and structural data, looking for a pattern of association of these molecules in the crystal structures and with the concept that ‘random things seldom repeat’ lead to a surprisingly interesting and consistent observation that (1) the weak cis-interaction mediated symmetric oligomerization of signaling molecules not only support their ‘upright’ orientation but often bury their ligand-binding surface to avoid spurious signaling (2) the linkers connecting the domains are probably not flexible as presumed. This analysis provides a model for pre-liganded receptor clustering that resolves some of the mysteries unanswered by hypothesis such as ‘lipid-rafts’ and ‘fence and pickets. With CD4, pMHCII, CD2 and TNFR1 as examples, we show that the observed cis-association of molecules also correlate well with their functional role and emphasise the need for considering these cis mediated physiological confirmations while designing the therapeutic agonist or antagonistic antibodies. Further, our analysis reconciles the long-standing controversies related to these molecules and appear to be generic enough to be applied to other signaling molecules. Finally, we also provide a new model for CD4:MHC:TCR signaling

Biological membranes surround all living cells, confining internal organelles and participating in a variety of essential cellular functions, such as signaling, electrolyte balance, and energy conversion. Cell membranes are structurally and chemically heterogeneous environment composed of numerous types of lipids arranged as a continuous bilayer. The assembly of protein complexes at the membrane surface is responsible for fundamental biological processes such as synaptic transmission, blood coagulation, and apoptosis. Resolving the macromolecular organization of these complexes at the membrane surface will help to understand the structural basis of their function and significance for the associated biological processes. In this review, we present our work on direct structure determination of membrane-bound clotting factors, specifically factor VIII (FVIII), by cryogenic electron microscopy (CryoEM). To resolve the FVIII membrane-bound organization, we have optimized lipid nanostructures resembling the activated platelet membrane. Combining structural CryoEM, capable of near-atomic resolution, with customized lipid nanotechnologies is a powerful approach to investigate how the cellular membrane can modulate protein function at close to physiological conditions. The outcome will open novel avenues for developing lipid nanotechnologies of diverse shapes and composition that can be optimized for various protein systems, germane for both drug delivery and macromolecular structure determination.

Oligomerization of hepatitis C viral envelope proteins E1 and E2 is essential to virus fusion and assembly. Although interactions within the transmembrane (TM) domains of these glycoproteins have proven contributions to the E1/E2 heterodimerization process and consequent infectivity, there is little structural information on this entry mechanism. Here, as a first step towards our long-term goal of understanding the interaction between E1 and E2 TM-domains, we have expressed, purified and characterized E1-TM using structural biomolecular NMR methods. An MBP-fusion expression system yielded sufficient quantities of pure E1-TM, which was solubilized in two membrane-mimicking environments, SDS- and LPPG-micelles, affording samples amenable to NMR studies. Triple resonance assignment experiments and relaxation measurements provided information on the secondary structure and global fold of E1-TM in these environments. In SDS micelles E1-TM adopts a helical conformation, with helical stretches at residues 354–363 and 371–379 separated by a more flexible segment of residues 364–370. In LPPG micelles a helical conformation was observed for residues 354–377 with greater flexibility in the 366–367 dyad, suggesting LPPG provides a more native environment for the peptide. Replacement of key positively charged residue K370 with an alanine did not affect the secondary structure of E1-TM but did change the relative positioning within the micelle of the two helices. These results lay the foundation for structure determination of E1-TM and a molecular understanding of how E1-TM flexibility enhances its interaction with E2-TM during heterodimerization and membrane fusion.

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.

This Review covers the delineation and optimization of protein–lipid systems for study using solution-state NMR spectroscopy. The first half presents the necessary background for a membrane protein biochemist to initiate collaboration with an NMR spectroscopist. The second half provides guidelines for the spectroscopist on data collection, analysis for obtaining conformational information, and structure generation and assessment. Although the emphasis is on the study of peptides in detergent micelles, methods are outlined for larger membrane-associated proteins and for use of other solubilizing agents.

Kv10.1 is voltage gated potassium channel of the EAG family, which is mainly expressed in the Central Nervous System (CNS) under physiological conditions. In the past years, a causal association between Kv10.1 and tumor growth has also been proven, nevertheless the precise mechanism by which this occurs remains unknown. In this work we compare the direct association of membrane-associated proteins to Kv10.1 in different membrane domains of brain and cancer cells. Plasma membranes of brain tissue of adult mice (C57BL/6) and of the human prostate cancer cell line DU-145 were isolated. Detergent resistant membranes (DRM) were extracted through Triton X-100 solubilization and discontinuous gradient centrifugation. IP-assay for Kv10.1 was performed in the different membrane domains; protein expression was analyzed through the Colloidal Coomasie method. Kv10.1 associates partially to the DRM in brain tissue whereas in cancer cells no association can be observed. The protein pattern associated to Kv10.1 in the different systems also varies. We conclude that the different domain association of Kv10.1 is important for different protein interactions and could be involved in it's role as a tumor promoting protein.

This chapter provides an introduction to the composition, structure and transport mechanisms of biological cellular and epithelial membranes. The type of lipids found in cell membranes, their chemical properties and their arrangement into a lipid bilayer are discussed as are the classes of membrane associated proteins and their characteristics. The third major component of biological membranes, carbohydrate, is also discussed as is its apparent functional role.

Solid phase peptide synthesis (SPPS) provides the possibility to chemically synthesize peptides and proteins. Applying the method on hydrophilic structures is usually without major drawbacks but faces extreme complications when it comes to "difficult sequences." These includes the vitally important, ubiquitously present and structurally demanding membrane proteins and their functional parts, such as ion channels, G-protein receptors, and other pore-forming structures. Standard synthetic and ligation protocols are not enough for a successful synthesis of these challenging sequences. In this review we highlight, summarize and evaluate the possibilities for synthetic production of "difficult sequences" by SPPS, native chemical ligation (NCL) and follow-up protocols.

View Large Image | View Hi-Res Image | Download PowerPoint SlideWe created two-dimensional (2D) assemblies of tobacco mosaic viruses (TMVs) and characterized their structures using Atomic Force Microscopy (AFM) and X-ray scattering. The TMVs were adsorbed on an oppositely charged, fluid lipid monolayer supported by a solid substrate and submerged in a buffer solution. The lipid monolayer confined the viral particles within a plane, while providing them with lateral mobility so that overall the TMV assembly behaved like a 2D liquid. The inter-particle interaction is controlled by the chemical condition in the buffer. The degree of structural orders observed varied, depending on both the inter-particle interaction and the lateral mobility of the particles. Quantitative analysis of the X-ray scattering data provides information on the nature of the interaction between TMVs as well as possible membrane deformation due to the contact with TMVs. This study provides the proof-of-concept that X-ray scattering may be used to study the structure of membrane associated proteins in substrate-supported single bilayer under near-native conditions.

Biological organisms generally maintain their lipid membrane composition so that the membranes are fluid at the growth temperature. This means that the lipids they contain can be moved by hydrodynamic flow in the fluid surrounding a cell [1]. Some organisms use flow deriving from their own movement to preferentially concentrate external proteins in the lipid membrane surface [2]. It has previously been shown that lipid-anchored proteins protruding from a supported lipid bilayer can be concentrated or dispersed by fluid flow applied to the membrane surface [3]. Here we investigate the effect of similar applied microfluidic flow on free, non-supported bilayers containing a low concentration of protruding proteins.References[1] Aurelia R. Honerkamp-Smith, Francis G. Woodhouse, Vasily Kantsler, and Raymond E. Goldstein. Membrane Viscosity Determined from Shear-driven Flow in Giant Vesicles. Physical Review Letters 111, p268102 (2013).[2] Markus Engstler, Thomas Pfohl, Stephan Herminghaus, Michael Boshart, Geert Wiegertjes, Niko Heddergott, Peter Overath. Hydrodynamic Flow-mediated Protein Sorting on the Cell Surface of Trypanosomes. Cell 131 (3), p505 (2007).[3] Peter Jonsson, James McColl, Richard W. Clarke, Victor P. Ostanin, Bengt Jonsson, and David Klenerman. Hydrodynamic trapping of molecules in lipid bilayers. Proceedings of the National Academy of Sciences of the United States of America 109 (26), p10328 (2012).

Our goal is to study interactions between bicelles and membrane associated proteins by computer simulations. In this preliminary study we have tested different representations of the bicelle lipids within the MARTINI coarse grain (CG) model. Bicelles are lipid disk-shaped systems with properties that are believed to resemble those of a native lipid bilayer. This has made them useful for studies of membrane associated proteins by a range of biophysical techniques, such as circular dichroism, liquid and solid state NMR and in diffraction studies. Bicelles normally consist of two different kinds of lipids, one with short hydrophobic tails and one with longer tails. The structure and composition of a bicelle depends on both the temperature and the ratio between the long and short tailed lipids, also known as the q-factor. The results from the CG molecular dynamics (MD) simulations are compared to NMR data, and by that revealing that this method can account for the experimental observations made of bicelle structures. Furthermore, reversed CG united atom MD simulations using the GROMOS96 force field indicate that the bicelles formed are stable on the time scale simulated. The results from the CG MD of bicelles also underline the importance of choosing a proper mapping of atoms to the CG beads.