Faculty Bibliography

Three-dimensional crystals were obtained for the membrane domain of the human erythrocyte anion exchanger (AE1, Band 3). Protein homogeneity and stability and the delicate balance between the detergent used and the amount of phospholipids copurifying are critical to the formation of three-dimensional crystals of the AE1 membrane domain. While deglycosylation improved the protein homogeneity, its stability was significantly increased by inhibitor binding. Size-exclusion chromatography showed that the protein was monodisperse in detergents with acyl chains of 10-12 carbons over a pH range of 5.5-10.0. This pH range and the detergents that retained the protein's monodispersity were used for crystallization screening. Crystals were obtained with the protein purified in C(12)E(8), dodecylmaltoside, decylthiomaltoside, and cyclohexyl-hexylmaltoside. Five to 13 lipid molecules per protein were required for the protein crystal formation. Those crystals grown in dodecylmaltoside diffracted X-rays to 14 A. With these factors taken into consideration, ways to further improve the crystal quality are suggested

The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.

The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 &mgr;M, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 &mgr;mol min(-)(1) mg(-)(1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-)(1) (mol of protein)(-)(1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter

The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter

ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membrane which extrudes arsenite or antimonite from the cytoplasm. The molecular weight of the subunit is 63 kDa. In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane. We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC. These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing. The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry. Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution. Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa. Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form

The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein consisting of a 43 kDa N-terminal cytosolic domain that binds the cytoskeleton, haemoglobin and glycolytic enzymes and a 52 kDa C-terminal membrane domain that mediates anion transport. Electron microscopy and three-dimensional image reconstruction of negatively stained two-dimensional crystals of the dimeric membrane domain revealed a U-shaped structure with dimensions of 60 x 110 A, and a thickness of 80 A. The structure is open on the top and at the sides, with the monomers in close contact at the base. The basal domain is 40 A thick and probably spans the lipid bilayer. The upper part of the dimer consists of two elongated protrusions measuring 25 x 80 A in projection, with a thickness of 40 A. The protrusions form the sides of a canyon, enclosing a wide space that narrows down and converges into a depression at the centre of the dimer on the top of the basal domain. This depression may represent the opening to a transport channel located at the dimer interface. Based on the available protein-chemical data, the two protrusions face the cytosolic side of the membrane and they appear to be dynamic

The structure of the light-harvesting chlorophyll a/b-protein complex, an integral membrane protein, has been determined at 3.4 A resolution by electron crystallography of two-dimensional crystals. Two of the three membrane-spanning alpha-helices are held together by ion pairs formed by charged residues that also serve as chlorophyll ligands. In the centre of the complex, chlorophyll a is in close contact with chlorophyll b for rapid energy transfer, and with two carotenoids that prevent the formation of toxic singlet oxygen

Two different thylakoid lipids are specifically associated with the light-harvesting complex of photosystem II (LHC-II). Digalactosyl diacyl glycerol (DGDG) binds to the isolated complex but can be removed by mild detergent treatment and anion-exchange chromatography. Removal of this lipid renders the complex unable to form two-dimensional or three-dimensional crystals. The ability to crystallize is completely restored by addition of pure DGDG, at a ratio of about four molecules per polypeptide for three-dimensional crystals, suggesting several binding sites at the periphery of the trimeric complex. Two-dimensional crystals of purified protein grown in the presence of DGDG are more highly ordered than those obtained from the unfractionated complex. The other lipid, phosphatidyl glycerol (PG), binds more firmly and cannot be removed with non-ionic detergent. Complete delipidation of LHC-II can be achieved either with phospholipase or by proteolytic cleavage of 49 amino acid residues at the N terminus. Both treatments dissociate the native, trimeric complex into monomers. This indicates that PG is directly involved in the formation of trimers, which are a prerequisite for two-dimensional and three-dimensional crystallization. Both lipids are therefore present in two-dimensional and three-dimensional crystals and have distinct roles in the structure of the complex