Membrane Lipid Environment Research Articles

Mechanism by which water and protein electrostatic interactions control proton transfer at the active site of channelrhodopsin.

Channelrhodopsins are light-sensitive ion channels whose reaction cycles involve conformation-coupled transfer of protons. Understanding how channelrhodopsins work is important for applications in optogenetics, where light activation of these proteins triggers changes in the transmembrane potential across excitable membranes. A fundamental open question is how the protein environment ensures that unproductive proton transfer from the retinal Schiff base to the nearby carboxylate counterion is avoided in the resting state of the channel. To address this question, we performed combined quantum mechanical/molecular mechanical proton transfer calculations with explicit treatment of the surrounding lipid membrane. The free energy profiles computed for proton transfer to the counterion, either via a direct jump or mediated by a water molecule, demonstrate that, when retinal is all-trans, water and protein electrostatic interactions largely favour the protonated retinal Schiff base state. We identified a conserved lysine group as an essential structural element for the proton transfer energetics in channelrhodopsins.

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments.

Mass spectrometry has emerged as an important structural biology tool for understanding membrane protein structure, function, and dynamics. Generally, structural mass spectrometry of membrane proteins has been performed on purified or reconstituted systems which lack the native lipid membrane and cellular environments. However, there has been progress in the use and adaptations of these methods for probing membrane proteins within increasingly more native contexts. In this Concept article the use and utility of structural mass spectrometry techniques for studying membrane proteins within native environments are highlighted.

Oligomeric Structure and Three-Dimensional Fold of the HIV gp41 Membrane-Proximal External Region and Transmembrane Domain in Phospholipid Bilayers.

The HIV-1 glycoprotein, gp41, mediates fusion of the virus lipid envelope with the target cell membrane during virus entry into cells. Despite extensive studies of this protein, inconsistent and contradictory structural information abounds in the literature about the C-terminal membrane-interacting region of gp41. This C-terminal region contains the membrane-proximal external region (MPER), which harbors the epitopes for four broadly neutralizing antibodies, and the transmembrane domain (TMD), which anchors the protein to the virus lipid envelope. Due to the difficulty of crystallizing and solubilizing the MPER-TMD, most structural studies of this functionally important domain were carried out using truncated peptides either in the absence of membrane-mimetic solvents or bound to detergents and lipid bicelles. To determine the structural architecture of the MPER-TMD in the native environment of lipid membranes, we have now carried out a solid-state NMR study of the full MPER-TMD segment bound to cholesterol-containing phospholipid bilayers. 13C chemical shifts indicate that the majority of the peptide is α-helical, except for the C-terminus of the TMD, which has moderate β-sheet character. Intermolecular 19F-19F distance measurements of singly fluorinated peptides indicate that the MPER-TMD is trimerized in the virus-envelope mimetic lipid membrane. Intramolecular 13C-19F distance measurements indicate the presence of a turn between the MPER helix and the TMD helix. This is supported by lipid-peptide and water-peptide 2D 1H-13C correlation spectra, which indicate that the MPER binds to the membrane surface whereas the TMD spans the bilayer. Together, these data indicate that full-length MPER-TMD assembles into a trimeric helix-turn-helix structure in lipid membranes. We propose that the turn between the MPER and TMD may be important for inducing membrane defects in concert with negative-curvature lipid components such as cholesterol and phosphatidylethanolamine, while the surface-bound MPER helix may interact with N-terminal segments of the protein during late stages of membrane fusion.

Control of Transmembrane Helix Dynamics by Interfacial Tryptophan Residues

Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment.

Pdr18 is involved in yeast response to acetic acid stress counteracting the decrease of plasma membrane ergosterol content and order

Saccharomyces cerevisiae has the ability to become less sensitive to a broad range of chemically and functionally unrelated cytotoxic compounds. Among multistress resistance mechanisms is the one mediated by plasma membrane efflux pump proteins belonging to the ABC superfamily, questionably proposed to enhance the kinetics of extrusion of all these compounds. This study provides new insights into the biological role and impact in yeast response to acetic acid stress of the multistress resistance determinant Pdr18 proposed to mediate ergosterol incorporation in plasma membrane. The described coordinated activation of the transcription of PDR18 and of several ergosterol biosynthetic genes (ERG2-4, ERG6, ERG24) during the period of adaptation to acetic acid inhibited growth provides further support to the involvement of Pdr18 in yeast response to maintain plasma membrane ergosterol content in stressed cells. Pdr18 role in ergosterol homeostasis helps the cell to counteract acetic acid-induced decrease of plasma membrane lipid order, increase of the non-specific membrane permeability and decrease of transmembrane electrochemical potential. Collectively, our results support the notion that Pdr18-mediated multistress resistance is closely linked to the status of plasma membrane lipid environment related with ergosterol content and the associated plasma membrane properties.

Perturbation of the yeast mitochondrial lipidome and associated membrane proteins following heterologous expression of Artemia-ANT

Heterologous expression is a landmark technique for studying a protein itself or its effect on the expression host, in which membrane-embedded proteins are a common choice. Yet, the impact of inserting a foreign protein to the lipid environment of host membranes, has never been addressed. Here we demonstrated that heterologous expression of the Artemia franciscana adenine nucleotide translocase (ANT) in yeasts altered lipidomic composition of their inner mitochondrial membranes. Along with this, activities of complex II, IV and ATP synthase, all membrane-embedded components, were significantly decreased while their expression levels remained unaffected. Although the results represent an individual case of expressing a crustacean protein in yeast inner mitochondrial membranes, it cannot be excluded that host lipidome alterations is a more widespread epiphenomenon, potentially biasing heterologous expression experiments. Finally, our results raise the possibility that not only lipids modulate protein function, but also membrane-embedded proteins modulate lipid composition, thus revealing a reciprocal mode of regulation for these two biomolecular entities.

Membrane lipid environment: Potential modulation of chemokine receptor function

Migration of leukocytes is typically mediated by G protein-coupled receptors (GPCRs) upon activation by specific ligands that range from small peptides, chemokines to a variety of lipidic molecules. The heptahelical receptors are highly dynamic structures and the signaling efficiency largely depends on the discrete contact with the ligand. In addition, several allosteric modulators of receptor activity have been reported, which do not induce migration by themselves. Another important mechanism modulating the activity of GPCRs is their local environment. Not only the membrane lipid composition influences the activity, but also direct binding of lipids, in particular cholesterol, was shown to alter receptor signaling properties. Recent findings indicate that also chemokine receptor activity is modulated by membrane lipids. In this short review we discuss this new paradigm and potential consequences for chemokine-induced migration.

Mechanisms by Which Lipids Shape the Reaction Coordinate of GlpG Protease

Rhomboid proteases are membrane-embedded enzymes whose catalytic activity depends on the composition of the lipid membrane that surrounds them. This makes rhomboids an excellent model system to dissect mechanisms by which lipids shape the reaction mechanisms of membrane proteins. Extensive all-atom molecular dynamics simulations of the Escherichia coli rhomboid, GlpG, indicate that the orientation of GlpG and lipid interactions at the substrate docking site, which are essential for substrate binding, depend on the lipid membrane composition. Different protein conformations can associate with different orientations in the membrane, suggesting that protein orientation is a dynamic parameter that changes during the reaction coordinate. The computations provide a comprehensive view of the molecular picture of GlpG/lipid interactions in different lipid membrane environments. This research was supported in part by funding from the Excellence Initiative of the German Federal and State Governments provided via the Freie Universität Berlin, and by an allocation of computing time from HLRN, the North-German Supercomputing Alliance (bec00076).

Effects of Channel Forming Peptides on Lipid Bilayer Dynamics and Leaflet Coupling

Altering the material properties of lipid bilayers has been shown to affect the biological activity of a number of channel-forming peptides and proteins. A prototypical example is gramicidin, in which the channel formation and lifetime directly depends on the lipid membrane in which it is embedded. However, less is known about how a membrane-spanning channel affects the lipid bilayer properties, particularly the elasticity. Here we use neutron spin echo spectroscopy (NSE) to measure both the collective bending and thickness fluctuation dynamics in dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers containing gramicidin at low peptide/lipid ratios. At these low concentrations, gramicidin incorporation did not have a measurable effect on the average bilayer structure but significantly impacted the collective membrane dynamics. The bending modulus of DMPC membranes increased with increasing gramicidin concentration, while the trend in DPPC was non-monotonic and first decreased at low gramicidin concentrations before increasing at higher concentrations. In contrast, the thickness fluctuation amplitude increased with increasing gramicidin concentration in both the DMPC and DPPC lipid bilayers. Notably, combining the bending and thickness fluctuation results revealed that the seemingly disparate concentration trends in the DMPC and DPPC lipid bilayers were both consistent with an increase in coupling between the bilayer leaflets with increasing gramicidin concentration. Together these results demonstrate that channel formation can have considerable effects on the surrounding lipid membrane elasticity and highlight the interplay between lipids and peptides in determining the membrane dynamics.

Glycosphingolipids-function and useage.

Our research concerns the synthesis and function of glycosphingolipids (GSLs) in eukaryotic cells. Despite extensive structural characterization of these sugar-lipid conjugates, defining their function is difficult due to poor aqueous solubility. The fact that no one gene encodes their synthesis and membrane GSL organization within micro domains or ‘rafts’, is dependent on both their carbohydrate and lipid composition, further complicate functional studies. Membrane GSLs often provide the interface between pathogenic microbes and/or their products and host cells, making GSL-based therapeutics a possible mode of infection control. In turn, microbial GSL-binding ligands provide probes to study GSL function. The GSL lipid moiety and membrane environment regulate GSL receptor function and likely, biosynthesis. We have made species which mimic some of these features. Understanding the basis of this ‘aglycone’ effect is a central focus. We have found cholesterol masking of GSL carbohydrate via conformational restriction of the GSL carbohydrate is an important feature of GSL receptor function. A H-Bond network results in the carbohydrate adopting a membrane parallel rather than membrane perpendicular conformation. This renders the carbohydrate less available for trans-ligand binding-e.g. antibodies (invisible GSLs). The carbohydrate now forms an ‘umbrella’ over the steroid OH, restricting the access of cholesterol binding ligands also. Cholesterol masking of GSLs is particularly important in tumour cells. The higher tumour cell cholesterol content results in the masking of the majority of tumour associated GSLs from antibody recognition and thereby plays an important role in tumour evasion of immune surveillance. Strategies to deplete tumour cholesterol should reverse this masking. Several MAbs in clinical antineoplastic use, target GSLs and tumour biopsy binding is increased by cholesterol depletion. We have worked extensively on the E.coli elaborated AB5 verotoxin-VT (aka Shiga toxin-Stx) and showed the B subunit binds to the GSL, globotriaosyl ceramide (Gb3). Gb3 is upregulated in many human tumours ovarian, breast, renal, gastric, pancreatic, brain, colon, lymphoma, and their neovasculature. Thus VT has both anti-tumour and anti angiogenic potential. We showed VT efficacy in a brain tumour xenograft model and this has been verified with many other tumour models. The ABC cassette transporter and multiple drug resistance pump, MDR1 (and some homologues) plays an important role in the trans membrane supply of precursor GSL for the synthesis of Gb3 (and other GSLs). This means that MDR1 overexpression in drug resistant tumours can lead to increased Gb3 content and thereby increased VT sensitivity. Following ligand binding, cell surface GSLs undergo internalization and a process of retrograde transport from endosomes to the trans Golgi network to the ER. GSL binding bacterial toxins e.g. VT, cholera toxin use this pathway to target the ER where the A subunit is translocated to the cytosol via hijacking the ER retro translocon used for ER associated degradation (ERAD) of partially misfolded nascent proteins. We have used this property as a means to temporarily competitively block ERAD in the genetic diseases in which ERAD eliminates the mutant protein and induce/exacerbate disease symptoms (e.g. F508delta CFTR cystic fibrosis, Gaucher disease). This GSL dependent blockade partially reverses disease symptoms in cell culture and animal disease models, and provides a potential therapy for many misfolded protein based genetic diseases. ERAD blockade is also an antineoplastic stratagem. The HIV adhesion, gp120 also binds GSLs, including Gb3. We identified Gb3 as a host resistance factor against HIV and are using this to design novel inhibitors of HIV infection.

Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations.

The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of β-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.

Dynamics of OmpF Trimer Formation in the Bacterial Outer Membrane of Escherichia coli.

The self-assembly of outer membrane protein F (OmpF) in the outer membrane of Escherichia coli Gram-negative bacteria was studied using multiscale molecular dynamics simulations. To accommodate the long time scale required for protein assembly, coarse-grained parametrization of E. coli outer membrane lipids was first developed. The OmpF monomers formed stable dimers at specific protein-protein interactions sites irrespective of the lipid membrane environment. The dimer intermediate was asymmetric but provided a template to form a symmetric trimer. Superposition analysis of the self-assembled trimer with the X-ray crystal structure of the trimer available in the protein data bank showed excellent agreement with global root-mean-square deviation of less than 2.2 Å. The free energy change associated with dimer formation was -26 ± 1 kcal mol-1, and for a dimer to bind to a monomer and to form a trimer yielded -56 ± 4 kcal mol-1. Based on thermodynamic data, an alternate path to trimer formation via interaction of two dimers is also presented.

Protein Sequence and Membrane Lipid Roles in the Activation Kinetics of Bovine and Human Rhodopsins

Rhodopsin is a G protein-coupled receptor found in the rod outer segments in the retina, which triggers a visual response under dim light conditions. Recently, a study of the late, microsecond-to-millisecond kinetics of photointermediates of the human and bovine rhodopsins in their native membranes revealed a complex, double-square mechanism of rhodopsin activation. In this kinetic scheme, the human rhodopsin exhibited more Schiff base deprotonation than bovine rhodopsin, which could arise from the ∼7% sequence difference between the two proteins, or from the difference between their membrane lipid environments. To differentiate between the effects of membrane and protein structure on the kinetics, the human and bovine rhodopsins were inserted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid nanodiscs and the kinetics of activation at 15°C and pH 8.7 was investigated by time-resolved absorption spectroscopy and global kinetic analysis. For both proteins, the kinetics in nanodiscs shows the characteristics observed in the native membranes, and is described by a multisquare model with Schiff base deprotonation at the lumirhodopsin I intermediate stage. The results indicate that the protein sequence controls the extent of Schiff base deprotonation and accumulation of intermediates, and thus plays the main role in the different activation kinetics observed between human and bovine rhodopsins. The membrane lipid does have a minor role by modulating the timing of the kinetics, with the nanodisc environment leading to an earlier Schiff base deprotonation.

A polar SxxS motif drives assembly of the transmembrane domains of Toll-like receptor 4

Like all members of the Toll-like receptor (TLR) family, TLR4 comprises of a large ectodomain (ECD) involved in ligand recognition at the cell-surface, and a cytosolic Toll/interleukin-1 receptor (TIR) signalling domain, linked by a lipid membrane-anchored transmembrane (TM) domain (TMD). Binding of immunostimulatory pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharide (LPS) to myeloid differentiation factor 2 (MD-2) coreceptor-complexed TLR4 leads to its dimerization, resulting in productive juxtaposition of TIR domains and the initiation of pro-inflammatory innate immune responses. Whilst the process of PAMP recognition is relatively well understood, thanks to numerous high-resolution crystallographic structures of ECDs, the mechanism by which such recognition is translated into TMD dimerization and activating conformational changes is less clear. Based on available biophysical and biochemical experimental data, ab initio modelling, and multiscale molecular dynamics (MD) simulations entailing a total of >13μs and >200μs of atomistic and coarse-grained sampling, respectively, we investigate the structural basis for TLR4 TMD dimerization within a biologically relevant lipid membrane environment. A key polar-xx-polar (637SxxS640) motif is shown to drive association of the TLR4 TMDs, and to maintain a flexible interface, which may be disrupted by selected point mutations. Furthermore, MD simulations of various TMD+ECD constructs have been used to investigate the coupling between domains, revealing that flexible linkers abrogate dimerization via aggregation of ECDs at the membrane surface, explaining previous biochemical observations. These results improve our understanding of the assembly and signalling mechanisms of TLR4, and pave the way for rational structure-based development of membrane-associated immunomodulatory molecules.

PIPERINE: A NATURAL BIOENHANCER

Herbal sources have been explored recently at very high frequency owing to their lower risk benefit ratio as compared to the modern allopathic medicine systems. Herbal bioenhancer is an agent of herbal origin or any phytomolecule, which is capable of enhancing bioavailability and bioefficacy of a particular drug or nutrient with which it is combined, without any typical pharmacological activity of its own at the dose used. The active compound of both Long pepper ( Piper longum ) and Black pepper ( Piper nigrum ) is piperine (1-piperoyl piperidine), which is responsible for bioenhancing effect. Piperine enhances absorption from gastrointestinal tract by various mechanisms and reduces gut metabolism of drugs. Piperine modulates membrane dynamics and lipid environment and increases permeability at site of absorption. Thus, piperine acts as absorption enhancer and is a potent inhibitor of drug metabolism as it inhibits various metabolizing enzymes like human p-glycoproteins, CYP3A4, UDP-glucose dehydrogenase (UDP-GDH), aryl hydrocarbon hydroxylase (AAH) and UDP-glucuronyl transferase. The current review also describes the mechanism of action of piperine and bioavailability action of piperine on drugs and nutrients.

Phytohormone Behavior in the Model Environment of Plant and Human Lipid Membranes.

Interactions between three auxins (indole-3-acetic acid (IAA), 2-naphthoxyacetic acid (BNOA), and 2,4-dichlorophenoxyacetic acid (2,4-D)) and model two-dimensional lipid systems mimicking plant and human cell membranes were investigated in monolayers formed at the air/water solution interface. The analysis was based on the recorded π-A isotherm characteristics complemented with Brewster angle microscopy. The influence of auxins on model membranes was discussed on the basis of condensation changes, modification of mutual lipid-lipid interactions in the mixed films, and morphological alteration of the surface domains on the microscopic scale. It was demonstrated that the lipid composition and mutual proportion of the artificial membranes together with sterol to main the phospholipid ratio play a crucial role in the context of auxin behavior in the membrane-mimicking environment. Apart from specific molecular interactions between studied phytohormones represented by auxins and lipids, the condensation of the investigated monolayers was found to be a regulative factor of model systems' susceptibility toward auxin action. Two effects were recognized: fluidizing of monolayers being in the liquid state (model membranes) and initialization of the three-dimensional structure formation in ordered sterol films at high surface pressure. The influence of auxin molecules on lipid interactions in the monolayer and diminishing of the film condensation was the largest for BNOA, due to the presence of the most bulky nonpolar, aromatic fragment in the molecule. It was also demonstrated that auxins interact with model plant membranes more selectively, stronger, and at markedly lower concentration than with the human membrane models.

Multiplexed Lipid Bilayers on Silica Microspheres for Analytical Screening Applications.

Most druggable targets are membrane components, including membrane proteins and soluble proteins that interact with ligands or receptors embedded in membranes. Current target-based screening and intermolecular interaction assays generally do not include the lipid membrane environment in presenting these targets, possibly altering their native structure and leading to misleading or incorrect results. To address this issue, an ideal assay involving membrane components would (1) mimic the natural membrane environment, (2) be amenable to high-throughput implementation, and (3) be easily multiplexed. In a step toward developing such an ideal target-based analytical assay for membrane components, we present fluorescently indexed multiplexed biomimetic membrane assays amenable to high-throughput flow cytometric detection. We build fluorescently multiplexed biomimetic membrane assays by using varying amounts of a fluorescently labeled lipid, NBD-DOPE [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)], incorporated into a phospholipid membrane bilayer supported on 3 μm silica microspheres. Using flow cytometry, we demonstrate this multiplexed approach by measuring specific affinity of two well-characterized systems, the fluorescently labeled soluble proteins cholera toxin B subunit-Alexa 647 and streptavidin-PE/Cy5, to membranes containing different amounts of ligand targets of these proteins, GM1 and biotin-DOPE, respectively. This work will enable future efforts in developing highly efficient biomimetic assays for interaction analysis and drug screening involving membrane components.

Role of Hydrophilic Groove of the TMEM16 Protein in Lipid Scrambling

The TMEM16 proteins are Ca2+-dependent ion channels and/or phospholipid scramblases that play key roles in a variety of physiological functions. Mutations in TMEM16F cause Scott syndrome, a bleeding disorder caused by impaired Ca2+-dependent externalization of phosphatidylserine (PS) in activated platelets, suggesting that this homologue functions as a scramblase. Explicit demonstration that some TMEM16 proteins are Ca2+-dependent phospholipid scramblases comes from reconstitution and assay of purified fungal TMEM16 homologs (afTMEM16 and nhTMEM16). The recently determined crystal structure of nhTMEM16, revealed a dimeric arrangement of the protein and the presence of hydrophilic groove on the opposite sides of the dimer interface exposed to the lipid membrane. Extensive mutagenesis studies have established the importance of the groove in the lipid scrambling by showing that mutations of specific residues in the groove region to either Trp or Ala significantly reduce the kinetics of lipid translocation. We have used a combination of unbiased and enhanced sampling atomistic molecular dynamics (MD) simulations of nhTMEM16 in lipid membrane and water environments to evaluate the structural context and detailed mechanisms for the measured changes in scrambling efficiency by mutant nhTMEM16 constructs. The findings center on the formation and mechanistic role of a continuous water pathway along the membrane-facing surface of the groove in the wild type protein that enables lipid translocation. The manner in which the mutations cause a significant reduction in the level of the hydration in the groove region points to a key role of the hydrophilic groove of TMEM16 in lipid scrambling.

Light-Harvesting Complex II in Controlled Architectures: Visualizing Changes in Fluorescence Lifetimes

In the first stages of photosynthesis, light-harvesting membrane protein complexes form an interconnected network, absorbing photons and transferring energy as electronic excited states with high efficiency. Light Harvesting Complex II (LHCII) has several important roles, including: (i) acting as an antenna complex for Photosystem II (PSII), (ii) activating Non-Photochemical Quenching (NPQ) and (iii) promoting multi-layer stacking of thylakoid membranes. In this study, we investigated how the optical properties of LHCII depend upon protein-protein and lipid-protein interactions. Microscale array patterns of either single- or multi-layers of LHCII were generated on solid substrates, and studied by atomic force microscopy and fluorescence microscopy with spectral and lifetime imaging. Fluorescence spectra confirmed that the native chromophore organization of LHCII was maintained. Interestingly, LHCII had lower fluorescence lifetimes in multi-layers, suggesting that increased LHCII-LHCII interactions promote the quenched state. In other experiments, LHCII was deposited onto solid supports at varying protein density and in the presence or absence of a surrounding lipid membrane. LHCII fluorescence was strongly enhanced when lipids were added, potentially due to decreased self-quenching as the lipids effectively dilute the LHCII membrane concentration, decreasing LHCII-LHCII interactions. These experiments reveal switching between quenched and unquenched states depending on LHCII-LHCII and LHCII-lipid interactions.

Role of Lipids in the Reaction Coordinate of GlpG Rhomboid Protease

Rhomboid proteases function in hydrated lipid membrane environments that can impact significantly the catalytic activity of the enzyme. How lipids participate in the reaction coordinate of rhomboid proteases is, however, unclear. We address this question by performing all-atom molecular dynamics simulations of the rhomboid protease from Escherichia coli, GlpG. We study GlpG starting from three different protein crystal structures, and explore interactions with lipids by considering five one-component lipid membranes with different lipid headgroups or alkyl chains, and in mixed membranes of phosphatidylethanolamine and phosphatidylgylcerol lipids. Lipid alkyl chains can visit inter-helical regions of the protein close to the gate helix, which can indicate regions of potential interest for substrate binding. Variability in how the protein can interact with lipids closest to the helical gate region suggests that membrane environments may impact proteolysis by participating in the substrate-binding step of the reaction coordinate.Research was supported in part by funding from the Excellence Initiative of the German Federal and State Governments via the Freie Universitat Berlin, and by an allocation of computing time from HLRN, The North-German Supercomputing Center (bec00076).