A defined protein–detergent–lipid complex for crystallization of integral membrane proteins: The cytochrome b ₆ f complex of oxygenic photosynthesis

Abstract

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 6f 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 Å spacing; R merge ‫؍‬ 8.8%

Characterization of the High-Spin Heme x in the Cytochrome b6f Complex of Oxygenic Photosynthesis

“…The top two pink bands in the SDS-PAGE gel ( Figure 3A), corresponding to cytochromes f and b 6 , respectively, were excised and soaked in a buffer containing 30 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 0.05% -D-undecyl maltoside overnight or until the gel strips became colorless. The light pinkish "soaking solution" was collected for determination of pyridine hemochrome spectra of covalently bound hemes.…”

“…In this study, visible and EPR spectra have been obtained of heme x in the cytochrome b 6 f complex prepared from the thermophilic cyanobacterium M. laminosus and spinach chloroplasts. The cytochrome b 6 f complex from both sources was shown in previous studies to be pure by mass spectroscopic analysis (5) and formation of crystals that diffract to high resolution (1,6). 6 f Complex.…”

“…The cytochrome b 6 f complex from both sources was shown in previous studies to be pure by mass spectroscopic analysis (5) and formation of crystals that diffract to high resolution (1,6). 6 f Complex. The cytochrome b 6 f complex from spinach chloroplasts and a thermophilic cyanobacterium, M. laminosus, was purified according to the method of Zhang et al (6)(7)(8).…”

“…Heme x was found in the site occupied by the ubiquinone molecule bound on the n-side of the cytochrome bc 1 complex in the mitochondrial respiratory chain. The single covalent thioether bond involves a conserved cysteine residue (Cys35) on the n-side (stromal side) of helix A of the cytochrome b 6 subunit of the b 6 f complex. Cys35 is not part of a signature heme binding sequence (-Cys-X-Y-Cys-His-) that is diagnostic for c-type hemes.…”

“…Now that the presence of the additional heme in the cytochrome b 6 f complex has been unequivocally demonstrated by an X-ray structure from both a cyanobacterium (1) and green alga (2), it is important to determine the spectral properties of this unique heme to obtain information about its ligation and oxidation state, which have implications for its function. In this study, visible and EPR spectra have been obtained of heme x in the cytochrome b 6 f complex prepared from the thermophilic cyanobacterium M. laminosus and spinach chloroplasts.…”