A defined protein-detergent-lipid complex for crystallization of integral membrane proteins: The cytochrome b6f complex of oxygenic photosynthesis.
The paucity of integral membrane protein structures creates a major bioinformatics gap, whose origin is the difficulty of crystallizing these detergent-solubilized proteins. The problem is particularly formidable for hetero-oligomeric integral membrane proteins, where crystallization is impeded by the heterogeneity and instability of the protein subunits and the small lateral pressure imposed by the detergent micelle envelope that surrounds the hydrophobic domain. In studies of the hetero (eight subunit)-dimeric 220,000 molecular weight cytochrome b(6)f complex, derived from the thermophilic cyanobacterium, Mastigocladus laminosus, crystals of the complex in an intact state could not be obtained from highly purified delipidated complex despite exhaustive screening. Crystals of proteolyzed complex could be obtained that grew very slowly and diffracted poorly. Addition to the purified lipid-depleted complex of a small amount of synthetic nonnative lipid, dioleolyl-phosphatidylcholine, resulted in a dramatic improvement in crystallization efficiency. Large crystals of the intact complex grew overnight, whose diffraction parameters are as follows: 94% complete at 3.40 A spacing; R(merge) = 8.8% (38.5%), space group, P6(1)22; and unit cell parameters, a = b = 156.3 A, c = 364.0 A, alpha = beta = 90 degrees, gamma = 120 degrees. It is proposed that the methodology of augmentation of a well-defined lipid-depleted integral membrane protein complex with synthetic nonnative lipid, which can provide conformational stability to the protein complex, may be of general use in the crystallization of integral membrane proteins.
Zhang
Huamin
H
Department of Biological Sciences, Lilly Hall of Life Sciences, Purdue University, West Lafayette, IN 47907-2054, USA.
Kurisu
Genji
G
Smith
Janet L
JL
Cramer
William A
WA
D003460:Crystallization; D018360:Crystallography, X-Ray; D000458:Cyanobacteria / Q000737:chemistry; D003573:Cytochrome b Group / Q000737:chemistry*; D045346:Cytochrome b6f Complex; D003902:Detergents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010788:Photosynthesis
D003573:Cytochrome b Group; D003902:Detergents; D008055:Lipids; D008565:Membrane Proteins; D045346:Cytochrome b6f Complex; D010100:Oxygen