flippant-An R package for the automated analysis of fluorescence-based scramblase assays.

BACKGROUND: The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps. RESULTS: With the extension package "flippant" to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results. CONCLUSIONS: "flippant" allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.

Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins.

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family show favorable behavior relative to conventional detergents, as manifested in multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family.

The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSLS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC synthases. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.

Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa.

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.

Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.

A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles.

A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of approximately 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.

An engineered opsin monomer scrambles phospholipids.

The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.

Light-independent phospholipid scramblase activity of bacteriorhodopsin from Halobacterium salinarum.

The retinylidene protein bacteriorhodopsin (BR) is a heptahelical light-dependent proton pump found in the purple membrane of the archaeon Halobacterium salinarum. We now show that when reconstituted into large unilamellar vesicles, purified BR trimers exhibit light-independent lipid scramblase activity, thereby facilitating transbilayer exchange of phospholipids between the leaflets of the vesicle membrane at a rate >10,000 per trimer per second. This activity is comparable to that of recently described scramblases including bovine rhodopsin and fungal TMEM16 proteins. Specificity tests reveal that BR scrambles fluorescent analogues of common phospholipids but does not transport a glycosylated diphosphate isoprenoid lipid. In silico analyses suggest that membrane-exposed polar residues in transmembrane helices 1 and 2 of BR may provide the molecular basis for lipid translocation by coordinating the polar head-groups of transiting phospholipids. Consistent with this possibility, extensive coarse-grained molecular dynamics simulations of a BR trimer in an explicit phospholipid membrane revealed water penetration along transmembrane helix 1 with the cooperation of a polar residue (Y147 in transmembrane helix 5) in the adjacent protomer. These results suggest that the lipid translocation pathway may lie at or near the interface of the protomers of a BR trimer.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants.

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations-F45L, V209M and F220C-yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Constitutive phospholipid scramblase activity of a G protein-coupled receptor.

Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homoeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. In addition, we demonstrate that ß2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modelling cell membranes.

Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD.

Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-ß-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence.

Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins.

The use of the Protemist XE, an automated discontinuous-batch protein synthesis robot, in cell-free translation is reported. The soluble Galdieria sulphuraria protein DCN1 was obtained in greater than 2mg total synthesis yield per mL of reaction mixture from the Protemist XE, and the structure was subsequently solved by X-ray crystallography using material from one 10 mL synthesis (PDB ID: 3KEV). The Protemist XE was also capable of membrane protein translation. Thus human sigma-1 receptor was translated in the presence of unilamellar liposomes and bacteriorhodopsin was translated directly into detergent micelles in the presence of all-trans-retinal. The versatility, ease of use, and compact size of the Protemist XE robot demonstrate its suitability for large-scale synthesis of many classes of proteins.

Cell-free protein synthesis technology in NMR high-throughput structure determination.

This chapter describes the current implementation of the cell-free translation platform developed at the Center for Eukaryotic Structural Genomics (CESG) and practical aspects of the production of stable isotope-labeled eukaryotic proteins for NMR structure determination. Protocols are reported for the use of wheat germ cell-free translation in small-scale screening for the level of total protein expression, the solubility of the expressed protein, and the success in purification as predictive indicators of the likelihood that a protein may be obtained in sufficient quantity and quality to initiate structural studies. In most circumstances, the small-scale reactions also produce sufficient protein to permit bioanalytical and functional characterizations. The protocols incorporate the use of robots specialized for small-scale cell-free translation, large-scale protein production, and automated purification of soluble, His(6)-tagged proteins. The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.

Cell-free translation of integral membrane proteins into unilamelar liposomes.

Wheat germ cell-free translation is shown to be an effective method to produce integral membrane proteins in the presence of unilamelar liposomes. In this chapter, we describe the expression vectors, preparation of mRNA, two types of cell-free translation reactions performed in the presence of liposomes, a simple and highly efficient purification of intact proteoliposomes using density gradient ultracentrifugation, and some of the types of characterization studies that are facilitated by this facile preparative approach. The in vitro transfer of newly translated, membrane proteins into liposomes compatible with direct measurements of their catalytic function is contrasted with existing approaches to extract membrane proteins from biological membranes using detergents and subsequently transfer them back to liposomes for functional studies.