The structure of the reaction center from Rhodobacter sphaeroides has been solved by using x-ray diffraction at a 2.55-Å resolution limit. Three lipid molecules that lie on the surface of the protein are resolved in the electron density maps. In addition to a cardiolipin that has previously been reported [McAuley, K. E., Fyfe, P. K., Ridge, J. P., Isaacs, N. W., Cogdell, R. J. & Jones, M. R. (1999) Proc. Natl. Acad. Sci. USA 96, 14706 -14711], two other major lipids of the cell membrane are found, a phosphatidylcholine and a glucosylgalactosyl diacylglycerol. The presence of these three lipids has been confirmed by laser mass spectroscopy. The lipids are located in the hydrophobic region of the protein surface and interact predominately with hydrophobic amino acids, in particular aromatic residues. Although the cardiolipin is over 15 Å from the cofactors, the other two lipids are in close contact with the cofactors and may contribute to the difference in energetics for the two branches of cofactors that is primarily responsible for the asymmetry of electron transfer. The glycolipid is 3.5 Å from the active bacteriochlorophyll monomer and shields this cofactor from the solvent in contrast to a much greater exposed surface evident for the inactive bacteriochlorophyll monomer. The phosphate atom of phosphatidylcholine is 6.5 Å from the inactive bacteriopheophytin, and the associated electrostatic interactions may contribute to electron transfer rates involving this cofactor. Overall, the lipids span a distance of Ϸ30 Å, which is consistent with a bilayer-like arrangement suggesting the presence of an ''inner shell'' of lipids around membrane proteins that is critical for membrane function.I ntegral membrane proteins are found in the cell membrane surrounded by lipids. The biophysical and biochemical properties of these proteins are critically determined by the lipid environment. The lipid composition of cell membranes is complex, containing a variety of phospholipids, glycolipids, and other small molecules. In the purple nonsulfur bacterium Rhodobacter sphaeroides, the major lipids are the phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, or diphosphatidyl glycerol, and two glycolipids, sulfoquinovosyl diacylglycerol and glucosylgalactosyl diacylglycerol (1). The relative amounts of these lipids vary significantly depending on specific growth conditions, in particular the oxygen level, although the fatty acid chains are always predominately 18:1 with only minor contributions from 18:0, 16:0, and 16:1 and trace amounts of smaller chains. The lipid composition is known to critically determine the morphology of the cell membrane, but whether the lipids can affect the photosynthetic energy conversion processes remains an outstanding question.Understanding the influence of the lipid properties on the photosynthetic complexes, or any other integral membrane proteins, is in most cases limited by the lack of detailed structural information concerning membrane proteins. To ...

The bacteriochlorophyll protein, or FMO protein, from Chlorobium tepidum, which serves as a light-harvesting complex and directs light energy from the chlorosomes attached to the cell membrane to the reaction center has been crystallized in a new space group. The crystals belong to the cubic space group P4(3)32 and the structure has been refined to a resolution 2.2 A with a R factor of 19.7%. The electron density maps show that the structure is composed of two beta sheets that surround seven bacteriochlorophylls as previously reported (Li et al. (1997) J Mol Biol 271: 456-471). The availability of the new data allows a more accurate refinement of the pigment-protein complex including identification of bound solvent molecules. Several structural differences probably contribute to the observed spectroscopic differences between the FMO proteins from Cb. tepidum and Prosthecochloris aestuarii, including differences in the planarity of corresponding tetrapyrroles. A citrate molecule is found on the surface of each protein subunit of the trimer from Cb. tepidum. However, the citrate molecule is over 15 A from any bacteriochlorophyll. The presence of the citrate probably does not contribute to the function of the protein although it does contribute to the crystallization as it interacts with a crystallographically related trimer. Among the 236 water molecules found in the protein are four that appear to play a special role in the properties of bacteriochlorophyll 2, as this tetrapyrrole is coordinated by one of these water molecules and the waters form a hydrogen-bonded network that leads to the surface of the protein.

The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70 degrees C small oligomers populate, showing a typical beta-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of approximately 12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of approximately 50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60 degrees C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ x mol(-1). Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.

Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganese-binding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highest-affinity site had a dissociation constant of approximately 1 microM. For the highest-affinity mutant, a first-order rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 muM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex.

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.