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PubMed2000

Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine.

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Bacteriorhodopsins↗
PubMedDate not supplied

Expression and characterization of a His-tagged 11S seed globulin from Amaranthus hypochondriacus in Escherichia coli.

DNA encoding a His-tagged 11S globulin from Amaranthus hypochondriacus (amarantin) was successfully expressed in Escherichia coli strains BL21 (DE3) and Origami (DE3). The two strains produced different accumulation patterns. Whereas most of the proamarantin expressed in BL21 (DE3) was localized in inclusion bodies, that produced in Origami (DE3) was soluble (76 mg/L). Sucrose density gradient ultracentrifugation analysis of the expressed soluble proamarantin revealed that the protein was assembled into trimers. Treatment of proamarantin trimers in vitro using purified asparaginyl endopeptidase resulted in the appearance of peptides of the sizes expected for acidic and basic chains. Because the proamarantin assembles into trimers with the expected sedimentation characteristics and is cleaved into acidic and basic chains rather than being degraded, the results suggest that the protein folding occurring in E. coli is similar to that taking place in seeds. The His-tagged proamarantin was purified in a single step by immobilized metal affinity chromatography with a final yield of 48 mg/L. The overexpression of proamarantin in E. coli, together with the one-step purification will facilitate further investigation of this storage protein through site-directed mutagenesis.

Allergens↗
PubMed2005

Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy.

Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.

Bacteriorhodopsins↗
PubMed1997

Adenosine 5'-triphosphate is required for the assembly of 11S seed proglobulins in vitro.

Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but mostly monomers were recovered at high translation rates. The prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated that ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated or when ATP was absent suggested that protein misfolding had occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten globular conformations and thereby compete for Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures.

Adenosine Triphosphate↗
PubMed2005

Individual subunits of the glutamate transporter EAAC1 homotrimer function independently of each other.

Glutamate transporters are thought to be assembled as trimers of identical subunits that line a central hole, possibly the permeation pathway for anions. Here, we have tested the effect of multimerization on the transporter function. To do so, we coexpressed EAAC1(WT) with the mutant transporter EAAC1(R446Q), which transports glutamine but not glutamate. Application of 50 microM glutamate or 50 microM glutamine to cells coexpressing similar numbers of both transporters resulted in anion currents of 165 and 130 pA, respectively. Application of both substrates at the same time generated an anion current of 297 pA, demonstrating that the currents catalyzed by the wild-type and mutant transporter subunits are purely additive. This result is unexpected for anion permeation through a central pore but could be explained by anion permeation through independently functioning subunits. To further test the subunit independence, we coexpressed EAAC1(WT) and EAAC1(H295K), a transporter with a 90-fold reduced glutamate affinity as compared to EAAC1(WT), and determined the glutamate concentration dependence of currents of the mixed transporter population. The data were consistent with two independent populations of transporters with apparent glutamate affinities similar to those of EAAC1(H295K) and EAAC1(WT), respectively. Finally, we coexpressed EAAC1(WT) with the pH-independent mutant transporter EAAC1(E373Q), showing two independent populations of transporters, one being pH-dependent and the other being pH-independent. In conclusion, we propose that EAAC1 assembles as trimers of identical subunits but that the individual subunits in the trimer function independently of each other.

Alanine↗
PubMed2002

Borg/septin interactions and the assembly of mammalian septin heterodimers, trimers, and filaments.

Septins constitute a family of guanine nucleotide-binding proteins that were first discovered in the yeast Saccharomyces cerevisiae but are also present in many other eukaryotes. In yeast they congregate at the bud neck and are required for cell division. Their function in metazoan cells is uncertain, but they have been implicated in exocytosis and cytokinesis. Septins have been purified from cells as hetero-oligomeric filaments, but their mechanism of assembly is unknown. Further studies have been limited by the difficulty in expressing functional septin proteins in bacteria. We now show that stable, soluble septin heterodimers can be produced by co-expression from bicistronic vectors in bacteria and that the co-expression of three septins results in their assembly into filaments. Pre-assembled dimers and trimers bind guanine nucleotide and show a slow GTPase activity. The assembly of a heterodimer from monomers in vitro is accompanied by GTP hydrolysis. Borg3, a downstream effector of the Cdc42 GTPase, binds specifically to a septin heterodimer composed of Sept6 and Sept7 and to the Sept2/6/7 trimer, but not to septin monomers or to other heterodimers. Septins associate through their C-terminal coiled-coil domains, and Borg3 appears to recognize the interface between these domains in Sept6 and Sept7.

Animals↗
PubMed1998

Membrane fusion mediated by baculovirus gp64 involves assembly of stable gp64 trimers into multiprotein aggregates.

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell-surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell-cell contacts, suggests that such multimeric complexes represent a fusion machine.

Animals↗
PubMed2006

Macroscopic hierarchical surface patterning of porphyrin trimers via self-assembly and dewetting.

The use of bottom-up approaches to construct patterned surfaces for technological applications is appealing, but to date is applicable to only relatively small areas (approximately 10 square micrometers). We constructed highly periodic patterns at macroscopic length scales, in the range of square millimeters, by combining self-assembly of disk-like porphyrin dyes with physical dewetting phenomena. The patterns consisted of equidistant 5-nanometer-wide lines spaced 0.5 to 1 micrometers apart, forming single porphyrin stacks containing millions of molecules, and were formed spontaneously upon drop-casting a solution of the molecules onto a mica surface. On glass, thicker lines are formed, which can be used to align liquid crystals in large domains of square millimeter size.

Journal Article↗
PubMed1990

Assembly of coronavirus spike protein into trimers and its role in epitope expression.

The folding and oligomerization of coronavirus spike protein were explored using a panel of monoclonal antibodies. Chemical cross-linking and sedimentation experiments showed that the spike of transmissible gastroenteritis virus is a homotrimer of the S membrane glycoprotein. The spike protein was synthesized as a 175,000-apparent-molecular-weight (175K) monomer subunit that is sensitive to endo-beta-N-acetylglucosaminidase H. Assembly of monomers into a trimeric structure was found to occur on a partially trimmed polypeptide and to be a rate-limiting step, since large amounts of monomers failed to trimerize 1 h after completion of synthesis. Terminal glycosylation of newly assembled trimers, resulting in the biosynthesis of three 220K oligomers, occurred with a half time of approximately 20 min. Monomeric (230K to 240K) processed forms were also observed in cells and in virions. The 175K monomeric form expressed four major antigenic sites previously localized within the amino-terminal half of the S polypeptide chain; however, two classes of trimer-restricted epitopes (borne by three 220K and/or three 175K oligomers) were identified. The S glycoprotein of coronavirus might be a valuable model system for discovering new aspects of the maturation of membrane glycoproteins.

Animals↗
PubMed2005

Octyl-decorated Fréchet-type dendrons: a general motif for visualisation of static and dynamic behaviour using scanning tunnelling microscopy?

A detailed STM study of monolayers of 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzaldehyde and 3,5-bis[(3,5-bisoctyloxyphenyl)methyloxy]benzyl alcohol adsorbed on graphite is presented. Very highly resolved scanning tunnelling microscopy images are observed at room temperature in air allowing the analysis of the conformation of the adsorbed molecules. These long-chain alkyl-decorated Fréchet-type dendrons are a powerful assembly motif and initially form a pattern based on trimeric units, assembled into hexagonal host structures with a pseudo-unit cell of seven molecules, one of which remains highly mobile. Over time, the supramolecular ordering changes from a trimeric into a dimeric pattern. The chirality arising from the adsorption onto a surface of the dendrons is discussed.

Journal Article↗
PubMed1998

The antigenic structure of the HIV gp120 envelope glycoprotein.

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.

Antibody Formation↗
PubMed1997

The role of the cystine loop in acetylcholine receptor assembly.

Nicotinic acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, and delta subunits, assembled into alpha2betagammadelta pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into alphabetagamma trimers, but the subsequent steps in assembly are blocked. When alpha subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when beta subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of alphabetagammadelta tetramers and before the addition of the second alpha subunit. After formation of the cystine loop, the alpha subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of alphabetagamma trimers and before the addition of the delta subunit. The data indicate that the alpha subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.

Amino Acid Sequence↗
PubMed2006

Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26.

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Binding Sites↗
PubMed1995

The folding and assembly pathway of tumour necrosis factor TNF alpha, a globular trimeric protein.

The mechanisms of folding and assembly of the globular, trimeric protein tumour necrosis factor-alpha (TNF) were studied by chemical cross-linking. This revealed the rapid accumulation of a dimeric intermediate. Under the conditions of renaturation used, formation of the trimer is complete within six minutes. The kinetics of change of intrinsic and 8-anilino-1-naphthalene sulfonic acid fluorescence are first order and, combined with the kinetics of association, reveal the presence of folding steps both before and subsequent to formation of the trimer. Results from gel exclusion chromatography and kinetics, together with the existence of an acid-induced molten globule, support the conclusion that TNF folds and assembles through a trimeric molten globule.

Anilino Naphthalenesulfonates↗
PubMed1985

Genetic analysis of subunit assembly of the tail fiber of bacteriophage T3.

Bacteriophage T3 virions have six tail fibers composed of the product of gene 17 (gp17). Each tail fiber is a trimer of gp17 polypeptide. To characterize the assembly process of the tail fiber, temperature-sensitive (ts) mutants of gene 17 (ts17) were analyzed by SDS-polyacrylamide gel electrophoresis and by extract complementation. Newly synthesized gp17 polypeptide chains matured to SDS-resistant native trimers with a half time of about 7.5 min at 30 degrees. Although all ts17 mutants had similar plating efficiencies at restrictive temperature (41.5 degrees or 42 degrees), they showed different phenotypes. tsNG75, whose mutation was located in the carboxyl-terminal region of gene 17, was defective in trimer assembly at 41.5 degrees. The ts tail fibers formed at 30 degrees lost the ability to attach to the tail upon treatment at 41.5 degrees. There was a change in temperature sensitivity of tsNG75 tail fibers upon attachment to the tail, suggesting that the tail fiber may change conformation after attachment to the tail. tsNG215 and tsNG169, whose mutation sites were located in the amino-terminal region of gene 17, were not defective in the trimer assembly and attachment to the tail at the restrictive temperature. tsNG215 tail fibers formed at 41.5 degrees appear to be aberrant because they were not active in extract complementation and their attachment to fiberless particles resulted in production of noninfectious phage. Tail fibers produced by cells infected with tsNG169 at the restrictive temperature were active in extract complementation. Phage particles were formed in tsNG169-infected cells at the restrictive temperature. These particles were infectious at the permissive temperature and the mutant was non-infectious only if infection was continued at the restrictive temperature. These phenotypic differences exhibited by different gene 17 mutants may indicate the regions within the gene 17 polypeptide that play a role(s) in the folding and assembly of gp17 and in the biological activity of the mature tail fiber.

Electrophoresis, Polyacrylamide Gel↗
PubMed2002

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins.

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.

Amino Acid Sequence↗