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 displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is 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 display favorable behavior relative to conventional detergents, as tested on 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.

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 homeostasis. 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. Additionally, we demonstrate that β2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modeling cell membranes.

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.

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.Although integral membrane proteins account for almost 25% of open reading frames in fully sequenced genomes, progress on understanding their structure and function has lagged behind their soluble counterparts. In part, this is due to the difficulty in obtaining sufficient quantities of homogenous protein for in vitro studies using traditional expression systems. For example, the available space in cellular membranes, the toxic effects of competition for the membrane insertion machinery, and incorrect lipid composition for proper folding may limit the utility of Escherichia coli for eukaryotic membrane protein production [1]. Efforts to study the enzymology and structure of membrane proteins have been hindered by these difficulties over several decades.As one example, stearoyl-CoA desaturases are integral membrane proteins thought to have four trans-membrane sequences [2]. They have a conserved motif consisting of 8 His residues hypothesized to provide at least some of the ligands to a catalytically essential diiron center [2]. In 1974, Strittmatter and colleagues published a preparation of the stearoyl-CoA desaturase from the livers of starved and then fed rats [3]. This achievement ultimately permitted a number of important properties of the enzyme complex to be elucidated [4][5][6]. However, no comparable reports on the successful purification of mammalian stearoyl-CoA desaturase have arisen in the ensuing four decades.

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. Sphingolipids (SLs)5 are ubiquitous in eukaryotic membranes, where they perform multiple functions. Besides acting as structural components of biological membranes, they participate in protein sorting and cell signaling via membrane rafts (1) and serve as critical apoptotic and anti-apoptotic second messengers. As such, sphingolipids have important influence in human disease (2). SLs also act as precursors of other essential lipid biosynthetic pathways, e.g. generation of ethanolamine phosphate for use in the Kennedy pathway (3) and subsequent synthesis of phospholipids and glycosylphosphatidyl inositols. The common SL synthesis pathway (Fig. 1) begins with the condensation of serine and palmitate by serine palmitoyl transferase to make the sphingoid base 3-ketodihydrosphinganine, which is then reduced to make sphinganine. Sphinganine is the acceptor for N-fatty acylation by ceramide synthase to make dihydroceramide, which is then desaturated to make ceramide. Ceramide synthase also utilizes sphingosine generated by ceramide turnover. Specific sphingolipid synthases (SLS) then transfer the polar head groups fr...