Thomas Wacker

Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose.

The X-ray structure of a sucrose-specific porin (ScrY) from Salmonella typhimurium has been determined by multiple isomorphous replacement at 2.4 Å resolution both in its uncomplexed form and with bound sucrose. ScrY is a noncrystallographic trimer of identical subunits, each with 413 structurally well-defined amino acids. A monomer is built up of 18 anti-parallel β-strands surrounding a hydrophilic pore, with a topology closely similar to that of maltoporin. Two non-overlapping sucrose-binding sites were identified in difference Fourier maps. The higher permeability for sucrose of ScrY as compared to maltoporin is mainly accounted for by differences in their pore-lining residues.

Crystallization of the photosynthetic light‐harvesting pigment‐protein complex B800‐850 of Rhodopseudomonas capsulata

The B800‐850 light‐harvesting complex of Rhodopseudomonas capsulata was crystallized in the presence of detergents. The crystals were obtained by a vapour diffusion technique, using polyethylene glycol as a precipitant. Crystals grew to a size of 0.5 × 0.5 × 0.3 mm within two weeks. Two different crystal forms were obtained; one is supposed to be triclinic, space group PI, the other orthorhombic, space group C2221. Both crystal forms diffract to approximately 1.0 nm. Absorption spectra and polyacrylamide gel electrophoresis demonstrate that all expected components, i.e. three polypeptides of apparent M r,8000, 10000 and 14000, bacteriochlorophyll a and carotenoids, are present and in native conformation.

A physical characterization of some detergents of potential use for membrane protein crystallization

Micellar solutions of lauryldimethylamine oxide, n‐dodecyl‐β‐D‐maltoside and 1‐dodecanoylpropanediol‐3‐phosphorylcholine were studied by use of small‐angle neutron scattering. These detergents have been selected due to their use as solubilizing agents for membrane proteins. LDAO was found to form a homogeneous, approximately spherical micelle with a radius of 20.7 Å and an M r of 16 000. N‐Dodecyl‐β‐D‐maltoside forms an inhomogeneous micelle with a core of low scattering density surrounded by a shell of high scattering density. The data are in accord with a micelle forming an oblate ellipsoid and the disaccharide group pointing outward radially from the hydrophobic group. The semi‐axes are 16.8 and 25.5 Å and the M r is 66 000. 1‐Dodecanoylpropanediol‐3‐phosphorylcholine forms a rather homogeneous, roughly spherical micelle. The radius is 24 Å, the M r being 28 700. The data indicate a tangential packing of the phosphorylcholine head groups into a polar layer of 3–4 Å surrounding the micelle core. The use of these detergents as solubilizing agents during membrane protein crystallization is discussed.

The influence of heptane-1,2,3-triol on the size and shape of LDAO micelles Implications for the crystallisation of membrane proteins

The presence of small amphiphiles has been found to be necessary in the crystallization of several membrane‐protein surfactant complexes. It has been suggested that the role of the small amphiphile may be to reduce the size of the surfactant belt around the protein, making the formation of crystals easier. Thus far it was not known if this would involve changes in micellar size in general or whether the small amphiphile would merely replace LDAO during crystal growth. In the present study we have used small angle neutron scattering to study mixed micelles of lauryldimethyl amine oxide (LDAO; hydrogenated and deuterated) and heptane‐1,2,3‐triol (HP). Our results show that with increasing overall HP concentrations mixed LDAO/HP micelles of decreasing mass and radius are formed. The composition of these micelles has been determined. HP thus may decrease the size of the surfactant belt around a protein before crystallisation by insertion into a host micelle. As HP is a ‘small amphiphile’ compared to the surfactants used for solubilization of membrane proteins, the curvature of the host micelle will be increased by its insertion.

Crystallization and preliminary X-ray analysis of porin from Rhodobacter capsulatus

Porin monomers of the phototrophic bacterium Rhodobacter capsulatus were purified. Crystals were obtained from a solution of porin solubilized with the detergent octyltetraoxyethylene within 5 days using the vapor phase equilibration technique. The crystals were rhombohedral with an edge length of 0.4 mm. The space group was trigonal R3 with unit cell constants of a=b=95 Å, c=147 Å. Reflexions were observed to 3.2 Å.

Crystallization and spectroscopic investigation with polarized light of the reaction center-B875 light-harvesting complex of Rhodopseudomonas palustris

A pigment‐protein complex containing a reaction center and the B875 light‐harvesting complex from Rhodopseudomonas palustris was purified and crystallized in the presence of detergent. Thin, rectangular crystals were obtained and used for optical spectroscopy. The protein pattern as well as the spectrum of the solubilized and crystallized complex show that the purified complex was crystallized without a major change in its composition and conformation. The absorption band intensities in the plane of the crystal as well as the spectra of the tilted crystal indicate that the 590 nm Qx transitions are aligned along the long axis of the crystal, while the 875 nm Qy transitions are aligned along the short axis of the crystal plane. The results favor a model where the antenna bacteriochlorophylls associated with one reaction center have a common Qx direction and perpendicular to it a distribution of 875 nm Qy transitions exhibiting a maximum along one direction.

Isolation of reaction center and antenna complexes from the halophilic purple bacterium Rhodospirillum salexigens. Crystallization and spectroscopic investigation of the B800–850 complex

Abstract Intracytoplasmic membranes were isolated from the halophilic phototrophic bacterium Rhodospirillum salexigens. The membrane fraction was solubilized in lauryldimethylamineoxide (LDAO) and the reaction center (RC) and antenna complexes were separated by sucrose gradient centrifugation and column chromatography on DEAE cellulose. The RC preparation contains three polypeptides of Mr similar to that of the RC of Rhodobacter sphaeroides. Two polypeptides, of apparent Mr 12000 and 10000, are associated with the B870 complex. The B800–850 complex absorbing at 800 and 837 nm, seems to contain two low-Mr polypeptides, of about Mr 9000. Crystals grown of the B800–850 complex showed X-ray reflections to 0.8 nm. Crystals of 10 × 30 μm edge length were used for microspectroscopy with polarized light. All absorption bands showed a strong dichroism in the plane of the rectangular platelet. The dichroism of the 800 nm band was stronger than that of the 837 nm band.

Crystallization and preliminary X-ray analysis of a peridinin-chlorophyll a protein from Amphidinium carterae

Crystals of a water‐soluble (M r ≈ 39 000) peridinin‐chlorophyll a protein from Amphidinium carterae are reported. The crystals diffract to 2.2 Å and belong to a monoclinic (B2) and a triclinic (P1) space group. Spectra of the protein in the crystal and in solution are almost identical.

Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of β-sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH2-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 μm and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.

Linear Dichroism (LD) and Absorption Spectra of Crystals of B800–850 Light-Harvesting Complexes of Rhodopseudomonas capsulata

The B800–850 light-harvesting complex of Rps. capsulata was crystallized in the presence of detergents. The crystals were obtained by vapour diffusion technique using polyethylene glycol 4000 (Merck, for gas chromatography). A technique to produce large and thick crystals suitable for X-ray diffraction is described elsewhere (1). For spectroscopic work, due to the high optical density of the crystals, thin extended crystalline plates are advantageous. The crystallization technique was therefore modified. The best yield of thin crystals was found when s-D-octylglucopyranoside (1% w/v) or octyltetraoxyethylene (1%) plus heptanetriol (3%) were used as detergents and sodium chloride (0.5 M) was present. Other conditions were similar to the ones given in (1). Similar crystallization methods were applied by us to the B800–850 complex of Rps. palustris, and small crystals were obtained. Crystallization of the B800–850 complex of Rps. spaeroides has also been reported by Allen et al. (see J.Allen, R.Taylor and G.Feher, this volume). Thin crystalline plates of the B800–850 complex of Rps. capsulata, along the shortest dimension, can have optical densities as low as 0.3 at 850 nm. They have the form of parallelograms with edges being 0.05 to 0.2 mm in length. These thin crystals have similar geometry to the triclinic ones used for X-ray diffraction (1). We therefore assume that both lattices are identical. Spectra were obtained both from thin crystals grown together with large crystals for X-ray studies and from thin crystals grown with the modified procedure as described above. Crystals were carefully pipetted with some mother liquor into a microcell, formed by two glass windows and a spacer of 20 urn thickness. The crystal was thus aligned with its largest surface parallel to the microcuvette walls.