1-2-oleoylphosphatidylcholine and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

We have recently reported the crystallization by reconstitution into lipid bilayer structures of Enzyme IIC(mtl), the transmembrane C-domain of the mannitol transporter from E. coli. The projected structure was determined to a resolution of 0.5 nm [J. Mol. Biol. 287 5 (1999) 845]. However, further investigation proved that these crystals were multilamellar stacks instead of 2D crystals, and therefore were unsuitable for three-dimensional structural analysis by electron crystallography. Understanding the crystallogenesis of these crystals could reveal the mechanism of formation of multilayers. In the present study, cryo-electron microscopy (cryo-EM) and turbidimetry are used to study the successive steps of reconstitution of Enzyme IIC(mtl) into phospholipid-containing structures and its crystallization under different conditions. Our experimental approach enabled us to distinguish the separate steps of reconstitution and crystallization. The salt concentration especially influenced the nature of the vesicles, either half open unilamellar or aggregated multilamellar, formed during reconstitution of Enzyme IIC(mtl). The presence of DOPE and DOPC and the temperature influenced the type of lipid structures that were formed during the crystallization phase of Enzyme IIC(mtl). Cryo-EM showed that protein crystallization is closely associated with the formation of isotropic lipid (cubic) phases. We believe that DOPE is responsible for the formation of these lipid cubic phases, and that crystallization is driven by exclusion of protein from these phases and its concentration into the lamellar phases. This mechanism is inextricably associated with the formation of multilayers.

Topics: Cholic Acids; Cryoelectron Microscopy; Crystallization; Detergents; Escherichia coli; Escherichia coli Proteins; Glucosides; Liposomes; Membrane Proteins; Models, Molecular; Nephelometry and Turbidimetry; Phosphatidylcholines; Phosphatidylethanolamines; Phosphoenolpyruvate Sugar Phosphotransferase System; Protein Structure, Tertiary; Temperature

Formation of a trans-complex between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25 drives membrane fusion. The structure of the core SNARE complex has been studied extensively. Here we have used atomic force microscopy to study the behavior of recombinant syntaxin 1A both in detergent extracts and in a lipid environment. Full-length syntaxin in detergent extracts had a marked tendency to aggregate, which was countered by addition of munc-18. In contrast, syntaxin lacking its transmembrane region was predominantly monomeric. Syntaxin could be integrated into liposomes, which formed lipid bilayers when deposited on a mica support. Supported bilayers were decorated with lipid vesicles in the presence, but not the absence, of full-length syntaxin, indicating that formation of syntaxin complexes in trans could mediate vesicle docking. Syntaxin complexes remained at the sites of docking following detergent solubilization of the lipids. Raised lipid domains could be seen in bilayers containing sphingomyelin, and these domains were devoid of syntaxin and docked vesicles in the presence, but not the absence, of cholesterol. Our results demonstrate that syntaxin is excluded from sphingomyelin-enriched domains in a cholesterol-dependent manner.

Topics: Animals; Antigens, Surface; Cholesterol; Cholic Acids; Detergents; Lipid Bilayers; Liposomes; Macromolecular Substances; Membrane Fusion; Membrane Microdomains; Microscopy, Atomic Force; Microscopy, Electron; Nerve Tissue Proteins; Particle Size; Peptide Fragments; Phosphatidylcholines; Protein Structure, Tertiary; Rats; Recombinant Fusion Proteins; Sphingomyelins; Syntaxin 1

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.

Topics: Bacteriorhodopsins; Cholic Acids; Dimyristoylphosphatidylcholine; Halobacterium salinarum; Lipid Bilayers; Membrane Proteins; Micelles; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipid Ethers; Pressure; Protein Folding; Protein Structure, Secondary; Spectrophotometry, Ultraviolet