PMID:15157613
| Citation |
Stuart, MC, Koning, RI, Oostergetel, GT and Brisson, A (2004) Mechanism of formation of multilayered 2D crystals of the enzyme IIC-mannitol transporter. Biochim. Biophys. Acta 1663:108-16 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1016/j.bbamem.2004.02.008 |
| Keywords |
Cholic Acids/chemistry; Cryoelectron Microscopy; Crystallization; Detergents/chemistry; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Glucosides/chemistry; Liposomes/chemistry; Membrane Proteins/chemistry; Models, Molecular; Nephelometry and Turbidimetry; Phosphatidylcholines/chemistry; Phosphatidylethanolamines/chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System/chemistry; Protein Structure, Tertiary; Temperature |
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