Membrane Lipid Environment Research Articles

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.

Photosystem II: its function, structure, and implications for artificial photosynthesis.

Somewhere in the region of 3 billion years ago an enzyme emerged which would dramatically change the chemical composition of our planet and set in motion an unprecedented explosion in biological activity. This enzyme used solar energy to power the thermodynamically and chemically demanding reaction of water splitting. In so doing it provided biology with an unlimited supply of hydrogen equivalents needed to convert carbon dioxide into the organic molecules of life. The enzyme, which facilitates this reaction and therefore underpins virtually all life on our planet, is known as Photosystem II (PSII). It is a multisubunit enzyme embedded in the lipid environment of the thylakoid membranes of plants, algae, and cyanobacteria. Over the past 10 years, crystal structures of a 700kDa cyanobacterial dimeric PSII complex have been reported with ever increasing improvement in resolution with the latest being at 1.9 Å. Thus the organizational and structural details of its many subunits and cofactors are now well understood. The water splitting site was revealed as a cluster of four Mn ions and a Ca ion surrounded by amino acid side chains, of which seven provide ligands to the metals. The metal cluster is organized as a cubane-like structure composed of three Mn ions and the Ca2+ linked by oxo-bonds with the fourth Mn attached to the cubane via one of its bridging oxygens together with another oxo bridge to a Mn ion of the cubane. The overall structure of the catalytic site is providing a framework on which to develop a mechanistic scheme for the water splitting process and gives a blue print and confidence for the development of catalysts for mimicking the reaction in an artificial photo-electrochemical system to generate solar fuels.

Novel Lipid Dynamics Around the Cytolysin-A Membrane-Pore Complex and its Intermediates

Pore forming toxins (PFTs) are ubiquitous weapons in the armoury of many organisms. It is often observed that small numbers of these potent proteins form stable pores, permeabilize the cell membrane and cause cell lysis. The complex role of the membrane in the formation of these pores, the influence of the pore on the structure and dynamics of the surrounding membrane and the expulsion of the central lipids upon pre-pore formation are important phenomena that are poorly understood due to the paucity of structural data.Multi-scale molecular dynamics was carried out on the PFT Cytolysin-A (ClyA), to address its interaction with the surrounding lipid membrane. A large heterogeneity in the lipid self-diffusivities were observed with the presence of more mobile and less mobile lipid fractions spanning the membrane. These results indicate that the local environment around the protein complex is markedly different from the rest of the membrane. Simulations of intermediate ClyA oligomers in a variety of membranes shows rapid evacuation of the central lipids from the interior to the free membrane surrounding the partially formed pore assembly. This implies that concerted lipid expulsion occurs prior to the formation of the dodecameric pore complex. This alternative hypothesis challenges the notion that destabilization and ejection of a membrane patch corresponding to the pore-lumen occurs after the pre-pore assembly on the membrane surface is complete. This mechanism could occur across PFT families and has implications on membrane resealing and cell recovery.

G-Protein-Coupled Receptor Activation Investigated using Small-Angle Neutron Scattering

G-protein-coupled receptors (GPCRs) represent the largest family of proteins in the human genome and comprise about 50% of current molecular drug targets. Rhodopsin is the GPCR involved in visual light perception and occurs naturally in a membrane lipid environment. Rhodopsin photoactivation yields cis-trans isomerization of retinal giving an equilibrium between inactive Meta-I and active Meta-II states. Here we address the question: does photoactivation lead to a single Meta-II conformation or is there an ensemble of substates described by an ensemble-activation mechanism (EAM) [1]? In this context small-angle neutron scattering (SANS) probes rhodopsin-detergent and rhodopsin-lipid complexes through measurement of the intensity of the neutrons scattered as a function of scattering vector I(q). Contrast variation enables us to highlight individual components of a multi-component system without isotopic labeling of the sample. Upon photoactivation, the Meta-I state was stabilized in CHAPS-solubilized rhodopsin, while Meta-II was trapped in DDM-solubilized rhodopsin. The ligand-free apoprotein opsin was obtained by photobleaching rhodopsin in the presence of hydroxylamine. The SANS spectra for the above rhodopsin substates were acquired from 80% D2O solutions and at contrast-matching points for both DDM and CHAPS samples. The data collected in 80% D2O samples provide structural information for both protein and detergent, while the data collected at contrast-matching points give information for the protein structure exclusively. Our experiments demonstrate that for detergent-solubilized rhodopsin, SANS with contrast variation can detect structural differences between the rhodopsin dark-state, Meta-I, Meta-II, and ligand-free opsin states. Dark-state rhodopsin is more conformationally flexible (less-compact) in DDM micelles compared to the CHAPS, which is consistent with an ensemble of activated Meta-II states. Furthermore, the time-dependent structural transitions between Meta-I and Meta-II as observed by time-resolved SANS will be crucial to understanding the ensemble-based activation. [1]A.V. Struts et al. (2011) NSMB18, 392-394.

Preferred Homo- and Hetero-Dimeric Configurations of All Major Opioid Receptor Subtypes as Predicted by Simulated Self-Association in Explicit Lipid-Water Environment

Recent crystallographic structures of G protein-coupled receptors, including members of the opioid receptor subfamily, have shown inter-protomer contacts between parallel orientations of the receptors within the crystal. Although it is tempting to speculate that these parallel arrangements are indicative of physiologically relevant dimeric configurations, it is unclear whether they would preferentially form within a lipid environment. We have performed a series of unbiased, coarse-grained molecular dynamics simulations to compare the ability of mu-, kappa-, and delta-opioid receptors (MOP, KOP and DOP, respectively) to freely associate into homo- or heteromeric configurations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol bilayer. Twenty-five independent system setups, each containing sixteen equally spaced and randomly oriented parallel receptors, were simulated for 10 microseconds each, to investigate spontaneous self-assembly of DOP, MOP, or KOP homomers, as well as DOP-MOP and DOP-KOP heteromers, using the MARTINI coarse-grained force field. From these 250 microseconds of simulation we have identified several common dimeric arrangements, predominantly involving specific combinations of transmembrane helices TM1, TM2, TM4, TM5 and TM6, and the juxtamembrane helix 8. Unlike the TM5/TM6 and TM1/TM2/H8 interfaces observed in the MOP crystal structure, the TM1/TM2/H8 interface presented by the KOP crystal structure was reliably reproduced during spontaneous association of the KOP, DOP, and DOP-KOP dimers within the lipid environment. MOP dimers did reproduce other crystallographic dimers sporadically, namely the TM1/TM2/H8 and TM5/TM6 interfaces from the beta2-adrenergic receptor and CXCR4 crystal structures, respectively. These extensive simulation data provide testable hypotheses of likely homo- and hetero-dimerization interfaces, as well as diffusion rates, for all major opioid receptor subtypes, contributing important insights into the nature of association of these receptors in a lipid membrane environment.

1P221 カリウムチャネルKcsAのゲート開閉と連動した膜中集合・離散(13C. 生体膜・人工膜:興奮・チャネル,ポスター,第52回日本生物物理学会年会(2014年度))

1P221 カリウムチャネルKcsAのゲート開閉と連動した膜中集合・離散(13C. 生体膜・人工膜:興奮・チャネル,ポスター,第52回日本生物物理学会年会(2014年度))

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

NMR-Restrained Protein Structure Calculations in Implicit Environment

The aqueous and lipid membrane environments that make up the intra- and extra-cellular compartments of biological organisms are critical for supporting the structural and functional integrity of both soluble proteins and membrane proteins. NMR spectroscopy is extremely versatile and adept at characterizing the structures and functions of both types of proteins in samples that closely resemble the native environment. However, structure calculations in explicit solvent or explicit lipids are computationally expensive. Therefore, even when NMR structural restraints are measured in a native-like protein environment, protein structures are typically calculated using simple repulsive potentials without physically meaningful terms for electrostatics, non-covalent interactions or atom solvation. To facilitate NMR structure calculations in the proper environment we are developing a computationally less demanding implicit solvent potential for the XPLOR-NIH NMR structure refinement package. Here we show that the potential provides significant improvements both in the quality and precision of the calculated structures; it improves the accuracy of structures determined with sparse restraints; it provides numerous physically meaningful hydrogen bonding connections; and it provides correct embedding of membrane proteins in lipid bilayer membranes.This research was supported by grants from the National Institutes of Health (AI074805; GM100265; GM094727; EB002031).

Investigation of Lipid Bilayer Effects on Rhodopsin Activation using UV-Visible and Ftir Spectroscopy

G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets and play crucial roles in signal transduction for a number of physiological processes. Upon photoactivation, rhodopsin undergoes a series of conformational changes leading to visual perception. An ensemble of activated Meta II states is in equilibrium with the inactive precursor Meta I [1]. Lipid bilayer composition and its interaction with membrane proteins govern the ensemble activation mechanism (EAM) as predicted by the flexible surface model (FSM). The FSM describes the balance between intrinsic monolayer curvature and lipid-protein hydrophobic interactions, leading to the elastic coupling of lipids and membrane proteins [2]. Effects of temperature and pH were analyzed for rhodopsin reconstituted in lipid vesicles using UV-visible and FTIR spectroscopy. Thermodynamic properties were derived by fitting phenomenological Henderson-Hasselbalch functions to their respective pH titration curves. Mixed-chain POPC membranes backshift rhodopsin towards Meta I, whereas rhodopsin in DOPC favors the active Meta IIa substate. Analysis of the wavelength-dependent distribution of pKa and alkaline endpoints as estimated from FTIR difference spectra reveals an ensemble of substates for each lipid bilayer-rhodopsin system. The presence of multiple activated conformations is a hallmark of the EAM. Our results are in agreement with the FSM, whereby lipids having a negative monolayer curvature favor the active Meta II state, while lipids with zero spontaneous curvature (POPC) favor the inactive Meta I state [3]. The data give additional insight into the entropy-enthalpy balance which drives the structural conformational changes that occur upon rhodopsin photoactivation. Moreover our work provides fundamental insight into the functionality of other GPCR-related proteins in a natural membrane lipid environment. [1] A.V. Struts (2011) PNAS 108, 8263-8268. [2] M.F. Brown (2012) Biochemistry 51, 9782-9795. [3] E. Zaitseva (2010) JACS 132, 4815-4821.

Protein Gymnastics in Thelipid Bilayer: Lipides as Determinants of Protein Structure

The orientation of transmembrane domains of polytopic membrane proteins with respect to the plane of the lipid bilayer is determined by a complex interplay between topogenic signals residing within the protein sequence, interaction of the protein with the translocon membrane insertion machinery, short-range and long-range interactions within the protein and the final environment of the protein largely determined by membrane lipid composition. Systematic alteration of lipid composition at steady state or dynamically after membrane protein assembly uncovered a role for lipid-protein interactions in determining initial membrane protein topogenesis as well as in dynamic topological re-organization after initial protein folding. Alteration of the charges within protein extramembrane domains coupled with changes in lipid composition demonstrated a synergistic and reciprocal relationship between protein and lipid charges in determining orientation of transmembrane domains. These results led to the Charge Balance Hypothesis, which posits that protein topogenic signals are decoded in accordance with positive-inside rule initially by the translocon but finally interpreted by electrostatic interactions between protein extramembrane domains and the membrane surface charge as determined by the collective membrane lipid head group composition. The steady state and dynamic nature of membrane protein organization observed in whole cells, as a function of the Charge Balance Hypothesis, has been faithfully reproduced with membrane proteins reconstituted in proteoliposomes thus establishing that such initial and dynamic protein organization is dependent solely on direct lipid-protein interactions independent of other cellular factors. Therefore, membrane protein topological organization can be viewed as highly dynamic rather than stable and static. The dynamic view of protein topological organization as influenced by the membrane lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded membrane proteins. Supported in part by NIH grant R37-GM20478.

Role of Membrane Lipids in Activating G-Protein-Coupled Receptors

The role of lipid-protein interactions in membrane function has long attracted the attention of researchers in the field of lipid membrane biophysics. Effects of membrane lipids on G-protein-coupled receptors (GPCRs) are revealed by UV-visible and FTIR spectroscopic studies of the conformational energetics of rhodopsin in visual signaling [1]. During rhodopsin photoactivation, the photoreactive 11-cis-retinylidene chromophore is isomerized to all-trans yielding an equilibrium between inactive Meta-I and active Meta-II states. Modulation of the metarhodopsin equilibrium depends on the polar head groups and the lipid acyl chain length and polyunsaturation. Membrane lipids can forward or back-shift the metarhodopsin equilibrium due to their chemically non-specific material properties [2]. A flexible surface model (FSM) describes elastic coupling of membrane lipids to the conformational energetics of rhodopsin. The new biomembrane model challenges the standard fluid mosaic model. Based on data first introduced for rhodopsin [2] the idea of a curvature stress field bridges theory and experiment. According to the FSM, membrane lipids whose spontaneous curvature stabilizes the activated state within the membrane are involved in regulating protein function. The new biomembrane model explains the effects of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress on visual signaling. An ensemble-mediated activation mechanism is proposed for rhodopsin in a natural membrane lipid environment, which includes a role for bulk water in the activation of rhodopsin-like GPCRs [4]. Ion channels, transporters, and membrane-bound peptides can all be affected by curvature forces due to elastic deformation of the bilayer, thus giving a new paradigm for membrane lipid-protein interactions in structural biology.[1] M.F. Brown (2012) Meth. Mol. Biol.914, 127-153.[2] M.F. Brown (1997) Curr. Top. Membr.44, 285-356.[3] M.F. Brown (2012) Biochemistry51, 9782-9795.[4] A.V. Struts (2011) PNAS 108, 8263-8268.

Stanley Opella – The conqueror of membrane protein structure

Proteins that span biological membranes perform some of the most important biochemical processes that are vital for life. Understanding their structural organisation is a prerequisite for developing drugs for treatment of diseases including various forms of cancer, cardiovascular disease, disorders of the immune and nervous systems etc. However, unravelling the detailed structural organisation of these proteins in their native membrane bound state has been virtually impossible. Amongst membrane proteins that are particularly important for drug development are G-protein-coupled receptors (GPCRs) which are a large family of membrane proteins. Approximately 50% of the currently available medications target this class of proteins. Detailed structural characterisation of these proteins is vital for developing drugs that can interact with specific binding sites and control their activities. Unfortunately, the most powerful technique for determining the three-dimensional structure of proteins, namely X-ray crystallography, cannot be readily used since membrane proteins are difficult to crystallise. To aid crystallisation of membrane proteins, it is necessary to remove the lipids that surround the protein, by adding detergents. Additional procedures used to obtain crystals include forming complexes with other proteins or even through insertions or deletions of entire protein domains within the GPCR protein. However, these manipulations could alter the structure of the GPCR which can hinder or slow down the process of designing drugs that require precise knowledge of the binding site within the intact protein molecule in its native lipid membrane environment. Consequently, one of the greatest challenge facing structural biologists is to be able to conquer the structure of membrane proteins without resorting to such manipulations which are often referred to as “divide and conquer”. The man who has been successful in meeting this challenge is Stanley Opella (Fig. 1) and his group at the University of California at San Diego [1]. They used solid state NMR spectroscopy to determine the complete three-dimensional structure of a GPCR protein, called CXCR1, intact in its membrane environment (Fig. 2). This is the first time the highresolution structure of such a large protein has been elucidated in its biologically relevant state. For this reason, Stanley Opella can be rightly described as the conqueror of membrane protein structure. This landmark achievement paves the way for determining the structure of not only other GPCRs but of other membrane proteins in their native membrane environment. Previously X-ray crystallography has been used to gain information on the structure of GPCR’s which helped to provide better insights into the mechanism of action of this class of proteins. The importance of this work was recognised through the award of a Nobel Prize in Chemistry to Robert Lefkowitz and Brian Kobilka [2,3]. The breakthrough made by Stanley Opella and his group will further advance our understanding of GPCRs and accelerate the development of life saving drugs.

Investigating the interactions of the 18 kDa translocator protein and its ligand PK11195 in planar lipid bilayers

The functional effects of a drug ligand may be due not only to an interaction with its membrane protein target, but also with the surrounding lipid membrane. We have investigated the interaction of a drug ligand, PK11195, with its primary protein target, the integral membrane 18kDa translocator protein (TSPO), and model membranes using Langmuir monolayers, quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectometry (NR). We found that PK11195 is incorporated into lipid monolayers and lipid bilayers, causing a decrease in lipid area/molecule and an increase in lipid bilayer rigidity. NR revealed that PK11195 is incorporated into the lipid chain region at a volume fraction of ~10%. We reconstituted isolated mouse TSPO into a lipid bilayer and studied its interaction with PK11195 using QCM-D, which revealed a larger than expected frequency response and indicated a possible conformational change of the protein. NR measurements revealed a TSPO surface coverage of 23% when immobilised to a modified surface via its polyhistidine tag, and a thickness of 51Å for the TSPO layer. These techniques allowed us to probe both the interaction of TSPO with PK11195, and PK11195 with model membranes. It is possible that previously reported TSPO-independent effects of PK11195 are due to incorporation into the lipid bilayer and alteration of its physical properties. There are also implications for the variable binding profiles observed for TSPO ligands, as drug–membrane interactions may contribute to the apparent affinity of TSPO ligands.

Media Effects on the Mechanism of Antioxidant Action of Silybin and 2,3-Dehydrosilybin: Role of the Enol Group

Silybin (SIL) and 2,3-dehydrosilybin (DHS) are constituents of milk thistle extract (silymarin) applied in the treatment of cirrhosis, hepatitis, and alcohol-induced liver disease. The molecular mechanism of their action is usually connected with antioxidant action. However, despite experimental and theoretical evidence for the antioxidant activity of SIL and DHS, the mechanism of their antiradical action still remains unclear. We studied the kinetics of SIL/DHS reactions with 2,2-diphenyl-1-picrylhydrazyl radical in organic solutions of different polarity and with peroxyl radicals in a micellar system mimicking the amphiphilic environment of lipid membranes. Kinetic studies together with determination of acidity and electrochemical measurements allowed us to discuss the structure-activity relationship in detail. In nonpolar solvents the reaction with free radicals proceeds via a one-step hydrogen atom transfer (HAT) mechanism, while significant acceleration of the reaction rates in methanol and water/methanol solutions suggests the dominating contribution of a sequential proton-loss electron-transfer (SPLET) mechanism with participation of the most acidic hydroxyl groups: 7-OH in SIL and 7-OH and 3-OH in DHS. In a heterogeneous water/lipid system, both mechanisms operate; however, the reaction kinetics and the antioxidant efficacy depend on the partition between lipid and water phases.

Structural and mechanistic principles of intramembrane proteolysis – lessons from rhomboids

Intramembrane proteases cleave membrane proteins in their transmembrane helices to regulate a wide range of biological processes. They catalyse hydrolytic reactions within the hydrophobic environment of lipid membranes where water is normally excluded. How? Do the different classes of intramembrane proteases share any mechanistic principles? In this review these questions will be discussed in view of the crystal structures of prokaryotic members of the three known catalytic types of intramembrane proteases published over the past 7 years. Rhomboids, the intramembrane serine proteases that are the best understood family, will be the initial area of focus, and the principles that have arisen from a number of structural and biochemical studies will be considered. The site-2 metalloprotease and GXGD-type aspartyl protease structures will then be discussed, with parallels drawn and differences highlighted between these enzymes and the rhomboids. Despite the significant advances achieved so far, to obtain a detailed understanding of the mechanism of any intramembrane protease, high-resolution structural information on the substrate-enzyme complex is required. This remains a major challenge for the field.

Electron Conduction and Photocurrent Generation of a Light-Harvesting/Reaction Center Core Complex in Lipid Membrane Environments.

To reveal the structure-function relationship of membrane proteins, a membrane environment is often used to establish a suitable platform for assembly, functioning, and measurements. The control of the orientation of membrane proteins is the main challenge. In this study, the electron conductivity and photocurrent of a light-harvesting/reaction center core complex (LH1-RC) embedded in a lipid membrane were measured using conductive atomic force microscopy (C-AFM) and photoelectrochemical analysis. AFM topographs showed that LH1-RC molecules were well-orientated, with their H-subunits toward the membrane surface. Rectified conductivity was observed in LH1-RC under precise control of the applied force on the probe electrode (<600 pN). LH1-RC embedded in a membrane generated photocurrent upon irradiation when assembled on an electrode. The observed action spectrum was consistent with the absorption spectrum of LH1-RC. The control of the orientation of LH1-RC by lipid membranes provided well-defined conductivity and photocurrent.

Detection and analysis of protofibrils and fibrils of hemoglobin: Implications for the pathogenesis and cure of heme loss related maladies

TFE induces structural alterations of proteins similar to the lipid environment of biological membranes, implicating these studies worthy of analyzing protein conformation in membranes such as red blood cells (RBCs). Heme loss occurs on rupturing of RBCs as found in diseases namely haemophilia, haemolytic anaemia, diabetes mellitus. TFE can be implied in discovering therapeutic targets, as it mimics the biological membrane environment. A global transition of hemoglobin (Hb) in presence of TFE was studied by using multi-methodological approach. The presence of partially folded state of Hb at 15% v/v TFE was confirmed by altered tryptophan environment, and retention of native-like secondary and tertiary structure. Molten globule state was observed at 20% v/v TFE as detected by increase tryptophan and high ANS fluorescence, slight alterations in Soret band relative to native. TFE on increasing concentration induced protofibrils at 25% v/v and fibrils at 45% v/v as depicted by altered tryptophan environment, heme loss, increase in non-native β-sheet secondary and tertiary structure, large hydrodynamic radii of heme-protein, high ANS, thioflavin T fluorescence and shift in Congo Red absorbance. Comet assay showed that protofibrils are cytotoxic to lymphocytes. SEM and XRD confirmed these aggregates to be fibrillar in nature.

Using crosslinkable diacetylene phospholipids to construct two-dimensional packed beds in supported lipid bilayer separation platforms

Separating and purifying cell membrane-associated biomolecules has been a challenge owing to their amphiphilic property. Taking these species out of their native lipid membrane environment usually results in biomolecule degradation. One of the new directions is to use supported lipid bilayer (SLB) platforms to separate the membrane species while they are protected in their native environment. Here we used a type of crosslinkable diacetylene phospholipids, diynePC (1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine), as a packed material to create a ‘two-dimensional (2D) packed bed’ in a SLB platform. After the diynePC SLB is exposed to UV light, some of the diynePC lipids in the SLB can crosslink and the non-crosslinked monomer lipids can be washed away, leaving a 2D porous solid matrix. We incorporated the lipid vesicle deposition method with a microfluidic device to pattern the location of the packed-bed region and the feed region with species to be separated in a SLB platform. Our atomic force microscopy result shows that the nano-scaled structure density of the ‘2D packed bed’ can be tuned by the UV dose applied to the diynePC membrane. When the model membrane biomolecules were forced to transport through the packed-bed region, their concentration front velocities were found to decrease linearly with the UV dose, indicating the successful creation of packed obstacles in these 2D lipid membrane separation platforms.

A Platform for Studying Structures of Membrane Proteins Inserted into Nanodiscs

Membrane proteins are critical to many biological processes and account for 70% of all know pharmacological targets. However, there are only a few hundred unique membrane protein structures available in protein data bank due to the difficulty in forming crystals for x-ray crystallography or electron crystallography. Several methods have been developed to study structures of membrane proteins in lipid membrane environments (e.g. helical, planar, or spherical membranes). Among those, insertion of membrane proteins in lipid nanodiscs offers some benefits in terms of size, homogeneity, and curvature. In the studied structures, the nanodisc is considered part of the whole complex. Here, we proposed to develop a platform to computationally remove the nanodisc contributions. This is essential for the proteins not inserted at a fixed position in nanodisc (i.e. proteins are floating in the nanodisc). In addition, the geometry of the nanodisc will be used to aid the determination of two of the three Euler angles. This will decrease the computational requirement, increase accuracy of the determined orientation of each individual protein particles, and thus increase the resolution of the determined structure.

Interaction of Local Anesthetics with Biomembranes Consisting of Phospholipids and Cholesterol: Mechanistic and Clinical Implications for Anesthetic and Cardiotoxic Effects

Despite a long history in medical and dental application, the molecular mechanism and precise site of action are still arguable for local anesthetics. Their effects are considered to be induced by acting on functional proteins, on membrane lipids, or on both. Local anesthetics primarily interact with sodium channels embedded in cell membranes to reduce the excitability of nerve cells and cardiomyocytes or produce a malfunction of the cardiovascular system. However, the membrane protein-interacting theory cannot explain all of the pharmacological and toxicological features of local anesthetics. The administered drug molecules must diffuse through the lipid barriers of nerve sheaths and penetrate into or across the lipid bilayers of cell membranes to reach the acting site on transmembrane proteins. Amphiphilic local anesthetics interact hydrophobically and electrostatically with lipid bilayers and modify their physicochemical property, with the direct inhibition of membrane functions, and with the resultant alteration of the membrane lipid environments surrounding transmembrane proteins and the subsequent protein conformational change, leading to the inhibition of channel functions. We review recent studies on the interaction of local anesthetics with biomembranes consisting of phospholipids and cholesterol. Understanding the membrane interactivity of local anesthetics would provide novel insights into their anesthetic and cardiotoxic effects.