The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 A resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 A in length, that runs along the sevenfold axis and ranges from 14 A to 46 A in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel beta barrel, to which each protomer contributes two beta strands, each 65 A long. The interior of the beta barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 A wide. The structure proves the heptameric subunit stoichiometry of the alpha-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of beta barrel pore-forming toxins.

Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal a-hemolysin (crHL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the aHlL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of acHL oligomers show a sevenfold axis of rotational symmetry. The aHiL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of aHL heteromers formed from subunits with the charge of wild-type aHL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the aHlL transmembrane pore both in three-dimensional crystals and on a biological membrane.As exemplified by studies on cholera toxin (1), chemical cross-linking and electron microscopy may not provide precise and unequivocal information on the subunit stoichiometry of oligomeric proteins. Complete cross-linking of an oligomeric protein can give both cyclic and linear species, which may migrate differently on denaturing polyacrylamide gels, giving the false indication of an additional subunit (1). Dynamic or static rotational disorder about the axis of molecular symmetry in two-dimensional crystals can yield misleading diffraction data (1). Resolution of the subunit composition of cholera toxin was achieved by the collection and analysis of high-resolution x-ray diffraction data (2). A second example is aerolysin, a pore-forming protein secreted by Aeromonas hydrophila. Aerolysin was thought to form a pentamer or a hexamer (3) until a recent examination by rotational correlation analysis of particles in averaged electron micrographs suggested that it is a heptamer (4). However, this conclusion is being reevaluated (5). The gap junction connexon provides another example of an oligomeric membrane protein for which the number of subunits is still unclear. The connexon was believed to be a hexamer (6), but recent work suggests that it may be a pentamer (7).Reservations concerning the reliability of methods employed in determining the subunit stoichiometry of other membrane proteins have led us to reexamine the quaternary structure of a-hemolysin (aHL), a protein that is a model system for studying membrane protein assembly (8-11), and that has potential applications in biotechnology [e.g., as a component of immunotoxins (12) or an element in biosensors (13)]. The aHL polypeptide of 293 amino acids is secreted by Staphylococcus aureus as a water-soluble monomer and assembles upon contact with lipid bilayers or the deterg...

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The cytochrome bel complex from bovine mitochonchia plays a central role in cell respiration. It is a membrane protein complex that consists of ten protein subunits and tom redox centers with a molecular weight of240,000 Daltons. Its crytallization has been repmted by several laboratmies. Ow· crystals, grown in the presence of glycerol, diffract Xray to better than 3 A resolution under cryogenic conditions. They have the symmel!Y of the spac;: group I4 1 22 with unit cell dime11sions of a=b=l 53 .S A and c=597. 7 A, and one bel complex in th;: crystallographic asymmetric unit. Pha<;es have been determined to 3.3 A resolution by the MIR method with fom heavy atom derivatives. The solvent flattened · electr·on density clearly shows the tr·ansmemebrane region with thirteen tr·ar1smembrane helices. Fom high peaks in the electr·on density ar1d in maps calculated using ar1omalous scattering data mea>umd near· the ir·on absorption edge ar·e ir1terpreted as the redox centers of the bel complex. Two of these sites are 20 A apart in the trar1smembrane region and most likely represent the heme ir·ons of cytochromes bs62 ar1d bs66· Another site near· the membrane smface ar1d 26 A away from the near-est b-heme could be the ir·on -sulf1.rr center~ the fomth site, presumably the cytochrome cl heme is 31 A apart from this center. The majmity of the molecular· mass outside the membrane is located on the side of the membrane opposite from the redox centers, presumably the matrix side of the mitochonchial membrar1e. The electr·on density map also reveals a very tight association of two monomers related by a two-fold symmetry. The overall dirnensions of the dimer ar-e about 130 A in diar-neter and 151 A in height, with the heights for tl1e inter-membrane space region, the tr·ansmembrane region ar1d t11e matri'\ region 41 A, 35 The reaction center of Photosystem II (Rem, a -250 kDa membrarle bound protein/pigment complex has been crystallized. RCII catalyzes the photochemically chiven transfer of electr·ons from water, resulting in tl1e fmmation of reduced and protonated quinones and the evolution of molecular-oxygen. It is tl1e somce of linear· electr·on t1ow utilized by all oxygenic photosyntl1etic organisms for botl1 the reduction ofNADP and the fom1ation of the proton gradient needed tor l\TP synt11esis. It is highly conserved in all species at all levels of str1.rct11re and function. Isolation procedmes have been developed for RCII from a variety of photosyntl1etic organisms witl1 an emphasis not only on pmity, but homogeneity and stability as well. Isolated RCII has been crystallized by the vapor diffusion method. Two Ciystal fon11s of isolated spinach RCII have been obtained: hexagonal rods and rectar1gular rods (with dir11en-sions of 0.3 x 0.3 x 1.0 mm). These Ciystals diffract to a maximum resolution of 7 A, using synchrotr·on radiation (SSRL Stanford). The crystals were however quite mosaic, appar-ently due to tl1eir sensitivity to physical str·ess. Botl1 crystal forms were obtained in tl1e presence of mixrures of two no...

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