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PubMed1996

The C-terminal domain of light-harvesting chlorophyll-a/b-binding protein is involved in the stabilisation of trimeric light-harvesting complex.

Light-harvesting chlorophyll a/b-binding protein (LHCP) can be reconstituted with pigments in detergent solution to yield stable monomeric light-harvesting chlorophyll a/b complex (LHCII). This reconstitution is not significantly affected when up to ten amino acids are deleted on the C-terminus of LHCP or when a tryptophan, which is 11 positions from the C terminus (W222), is exchanged with other amino acids [Paulsen, H. & Kuttkat, A. (1993) Photochem. Photobiol. 57, 139-142]. Here we show that the exchange of W222 with histidine or glycine completely abolishes the ability of the protein to assemble into trimeric LHCII, either upon reconstitution of monomeric complexes in detergent solution or upon insertion into isolated thylakoids. It is concluded that part of the hydrophilic domain on the C-terminus of LHCP, although not essential for the formation of stable monomeric LHCII, is involved in trimer formation. The different degree to which various amino acids in place of W222 affect trainer formation suggests that a hydrophobic amino acid is needed in this position.

Amino Acid Sequence↗
PubMed2006

Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography.

In the last few years, SAXS of biological materials has been rapidly evolving and promises to move structural analysis to a new level. Recent innovations in SAXS data analysis allow ab initio shape predictions of proteins in solution. Furthermore, experimental scattering data can be compared to calculated scattering curves from the growing data base of solved structures and also identify aggregation and unfolded proteins. Combining SAXS results with atomic resolution structures enables detailed characterizations in solution of mass, radius, conformations, assembly, and shape changes associated with protein folding and functions. SAXS can efficiently reveal the spatial organization of protein domains, including domains missing from or disordered in known crystal structures, and establish cofactor or substrate-induced conformational changes. For flexible domains or unstructured regions that are not amenable for study by many other structural techniques, SAXS provides a unique technology. Here, we present SAXS shape predictions for PCNA that accurately predict a trimeric ring assembly and for a full-length DNA repair glycosylase with a large unstructured region. These new results in combination with illustrative published data show how SAXS combined with high resolution crystal structures efficiently establishes architectures, assemblies, conformations, and unstructured regions for proteins and protein complexes in solution.

Crystallography, X-Ray↗
PubMed2003

Characterisation of Drosophila thrombospondin defines an early origin of pentameric thrombospondins.

Thrombospondins (TSPs) are multidomain oligomers that have complex roles in cell interactions and tissue organisation. The five vertebrate TSPs comprise two subgroups, A and B, that are assembled as trimers or pentamers, respectively. An invertebrate TSP was recently discovered in Drosophila melanogaster, but there is no knowledge of the oligomerisation status or properties of this molecule. We developed by bioinformatics a new dataset containing the single TSP of Drosophila melanogaster and four other newly identified invertebrate TSPs to examine the phylogenetic relationships of TSPs. These analyses clearly indicate pentamerisation as an early attribute of TSPs. We demonstrate experimentally that D.melanogaster TSP is assembled as a pentamer, has heparin-binding activity and is a component of extracellular matrix (ECM). During embryogenesis, the TSP transcript is concentrated at muscle attachment sites and is expressed by a subset of myoblasts and in imaginal discs. These novel results establish TSPs as highly conserved ECM components in both invertebrates and vertebrates and open fresh perspectives on the conservation of structure and biological function within this family.

Amino Acid Sequence↗
PubMed1995

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Base Sequence↗
PubMed2004

A trimeric quaternary structure is conserved in bacterial and human glutamate transporters.

Neuronal and glial glutamate transporters play a central role in the termination of synaptic transmission and in extracellular glutamate homeostasis in the mammalian central nervous system. They are known to be multimers; however, the number of subunits forming a functional transporter is controversial. We studied the subunit stoichiometry of two distantly related glutamate transporters, the human glial glutamate transporter hEAAT2 and a bacterial glutamate transporter from Escherichia coli, ecgltP. Using blue native polyacrylamide gel electrophoresis, analysis of concatenated transporters, and chemical cross-linking, we demonstrated that human and prokaryotic glutamate transporters expressed in Xenopus laevis oocytes or in mammalian cells are assembled as trimers composed of three identical subunits. In an inducible mammalian cell line expressing hEAAT2 the glutamate uptake currents correlate to the amount of trimeric transporters. Overexpression and purification of ecgltP in E. coli resulted in a homogenous population of trimeric transporters that were functional after reconstitution in lipid vesicles. Our results indicate that an evolutionarily conserved trimeric quaternary structure represents the sole native and functional state of glutamate transporters.

Amino Acid Sequence↗
PubMed2003

Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae of the order NIDOVIRALES: Six transmembrane proteins have been identified in EAV particles: the nonglycosylated membrane protein M and the glycoprotein GP(5) (previously named G(L)), which occur as disulfide-bonded heterodimers and are the major viral envelope proteins; the unglycosylated small envelope protein E; and the minor glycoproteins GP(2b) (formerly designated G(S)), GP(3), and GP(4). Analysis of the appearance of the GP(2b), GP(3), and GP(4) proteins in viral particles by gel electrophoresis under reducing and nonreducing conditions revealed the occurrence of two different covalently linked oligomeric complexes between these proteins, i.e., heterodimers of GP(2b) and GP(4) and heterotrimers of GP(2b), GP(3), and GP(4). Shortly after their release from infected cells, virions contained mainly cystine-linked GP(2b)/GP(4) heterodimers, which were subsequently converted into disulfide-bonded GP(2b)/GP(3)/GP(4) trimers through the covalent recruitment of GP(3). This process occurred faster at a higher pH but was arrested at 4 degrees C. Furthermore, the conversion was almost instantaneous in the presence of the thiol oxidant diamide. In contrast, the sulfhydryl-modifying agent N-ethylmaleimide inhibited the formation of disulfide-bonded GP(2b)/GP(3)/GP(4) trimers. Using sucrose density gradients, we could not demonstrate a noncovalent association of GP(3) with the cystine-linked GP(2b)/GP(4) dimer in freshly released virions, nor did we observe higher-order structures of the GP(2b)/GP(4) or GP(2b)/GP(3)/GP(4) complexes. Nevertheless, the instantaneous diamide-induced formation of disulfide-bonded GP(2b)/GP(3)/GP(4) heterotrimers at 4 degrees C suggests that the three minor glycoproteins of EAV are assembled as trimeric complexes. The existence of a noncovalent interaction between the cystine-linked GP(2b)/GP(4) dimer and GP(3) was also inferred from coexpression experiments showing that the presence of GP(3) increased the electrophoretic mobility of the disulfide-bonded GP(2b)/GP(4) dimers. Our study reveals that the minor envelope proteins of arteriviruses enter into both covalent and noncovalent interactions, the function of which has yet to be established.

Animals↗
PubMed2002

The N terminus of the MUC2 mucin forms trimers that are held together within a trypsin-resistant core fragment.

The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.

Amino Acid Sequence↗
PubMed2006

Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans.

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.

Chemical Phenomena↗
PubMed2002

Hantavirus nucleocapsid protein coiled-coil domains.

The nucleocapsid (N) proteins of hantaviruses such as the Sin Nombre virus (SNV) bind to membranes and viral RNAs, associate with transcription and replication complexes, and oligomerize during the process of virus assembly. N proteins trimerize in vitro and in vivo, and associate via assembly domains at their amino- and carboxyl-terminal ends. Because structure prediction algorithms suggested that N protein residues 3-75 form two coiled-coil motifs separated by an intervening kink or turn sequence, we examined the properties of peptides representing SNV N protein residues 3-35, 43-75, and 3-75. Of the three peptides, N-(3-35) assembled coiled-coil oligomers only at high concentration and low temperature. In contrast, N-(43-75) efficiently trimerized at low concentration, implying that it carries a coiled-coil trigger sequence. Interestingly, while the longer peptide, N-(3-75), assembled dimers and/or trimers at high concentration, at low concentration it appeared to adopt an intramolecular helix-turn-helix conformation. These results suggest that N protein oligomerization involves the bundling of intramolecular antiparallel coils or a conformational switch from intra- to intermolecular coiled-coils.

Amino Acid Motifs↗
PubMed2005

Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.

Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.

Animals↗
PubMed1999

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.

Amino Acid Sequence↗
PubMed1996

Mutation in the carboxy-terminal propeptide of the Pro alpha 1(I) chain of type I collagen in a child with severe osteogenesis imperfecta (OI type III): possible implications for protein folding.

A young girl presented with severe type III osteogenesis imperfecta; her otherwise healthy mother also had a mild connective tissue disorder with blue sclerae and recurrent joint dislocations. Skin fibroblast cultures from the child produced both normal and post-translationally over-modified type I collagen. The mutant collagen was poorly secreted but had normal thermal stability. Cyanogen bromide peptide maps of the abnormal protein indicated a C-terminal mutation. The mother's cells produced only normal-appearing collagens. Mismatch analysis and extensive sequencing of cDNAs covering the suspect region did not reveal any potentially causal changes in the triple helical domains of either the alpha 1(I) or alpha 2(I) chains. However, examination of the C-propeptide sequences revealed two heterozygous single base changes in the child. One, an A->C changing threonine to proline at residue 29 of the alpha 2(I) C-propeptide was also present in the mother and maternal grandfather but not in 50 unrelated control individuals. The second, a T->C altered the last amino acid residue of the alpha 1(I) C-propeptide from leucine to proline and had occurred de novo in the affected child. This mutation highlights the importance of the C-propeptides in molecular assembly but it is not clear how such an extreme mutation causes the delay in triple helix formation indicated by the extensive over-modification and reduced secretion of the mutant type I collagen. It may inhibit intrachain disulfide bonding or possibly affect the association of the procollagen chain with an intracellular "chaperone" protein that normally assists the assembly of trimeric procollagen molecules.

Amino Acid Sequence↗
PubMed1988

Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa.

Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1-3). These comprise 30%, 10%, and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr approximately 150,000) composed of polypeptides ranging in size from Mr 14,000 to 50,000 (alpha 1-alpha 6; 50,000, 38,000, 32,000, 20,000, 16,000 and 14,000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1-A9; Mr 49,000 to 39,000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24,000, 23,000 and 20,000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1/B2). Subunits (Mr approximately 50,000-65,000) are assembled as trimers (8S) or larger oligomers (12S-15S) in mature seeds. The lower molecular weight albumins (LMW1-3) are acidic (pI less than 6), and consist of sets of disulphide-bonded polypeptides (Mr 15,000 and 11,000).

Amino Acids↗
PubMed1996

Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase.

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.

Amino Acid Sequence↗
PubMed2000

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta.

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.

Blotting, Western↗
PubMed1996

Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL).

An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that it may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders.

Adult↗
PubMed1999

Two-dimensional crystallization of Escherichia coli lactose permease.

A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-dimensional (2D) crystals as shown by electron microscopy of negatively stained specimens. Single-particle analysis of 16 383 protein particles in densely packed vesicles reveals a 5.4-nm-long trapeziform protein of 4.1 to 5.1 nm width, with a central stain-filled indentation. Depending on reconstitution conditions, trigonal and rectangular crystallographic packing arrangements of these elongated particles assembled into trimers are observed. The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection maps calculated to a resolution of 2 nm show that these crystals consist of two layers.

Carrier Proteins↗