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| Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins |
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| Author(s): Levy D, Mosser G, Lambert O, Moeck GS, Bald D, Rigaud JL |
| Source: JOURNAL OF STRUCTURAL BIOLOGY Volume: 127 Issue: 1 Pages: 44-52 Published: AUG 1999 |
| Times Cited: 42 References: 38 |
| Abstract: A considerable interest exists currently in designing innovative strategies to produce two-dimensional crystals of membrane proteins that are amenable to structural analysis by electron crystallography. We have developed a protocol for crystallizing membrane protein that is derived from the classical lipid-layer two-dimensional crystallization at the air/water interface used so far for soluble proteins. Lipid derivatized with a Ni2+-chelating head group provided a general approach to crystallizing histidine-tagged transmembrane proteins. The processes of protein binding and two-dimensional crystallization were analyzed by electron microscopy, using two prototypic membrane proteins: FhuA, a high-affinity receptor from the outer membrane of Escherichia coli, and the F0F1-ATP synthase from thermophilic Bacillus PS3. Conditions were found to avoid solubilization of the lipid layer by the detergent present with the purified membrane proteins and thus to allow binding of micellar proteins to the functionalized lipid head groups. After detergent removal using polystyrene beads, membrane sheets of several hundreds of square micrometers were reconstituted at the interface. High protein density in these membrane sheets allowed further formation of planar two-dimensional crystals. We believe that this strategy represents a new promising alternative to conventional dialysis methods for membrane protein 2D crystallization, with the additional advantage of necessitating little purified protein. (C) 1999 Academic Press. |
| Document Type: Article |
| Language: English |
| Reprint Address: Levy, D (reprint author), Inst Curie, CNRS, UMR 168, Sect Rech, 11 Rue P&M Curie, F-75231 Paris, France |
Addresses:
1. Inst Curie, CNRS, UMR 168, Sect Rech, F-75231 Paris, France
2. CEA 8, LRC, F-75231 Paris, France
3. Univ Paris Sud, CNRS, ERS 571, Lab Biomembranes, F-91405 Orsay, France
4. Tokyo Inst Technol, Resources Utilizat Res Lab, CREST, Team 13,Midori Ku, Yokohama, Kanagawa 227 Japan |
| Publisher: ACADEMIC PRESS INC, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA |
| Subject Category: Biochemistry & Molecular Biology; Biophysics; Cell Biology |
| IDS Number: 240DG |
| ISSN: 1047-8477 |
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