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PubMed1993

GroEL, GroES, and ATP-dependent folding and spontaneous assembly of ornithine transcarbamylase.

When purified rat liver ornithine transcarbamylase (OTC), a trimer of 36 kDa subunits, was denatured in 6 M guanidine hydrochloride and then diluted 50-100-fold, no activity was recovered, and the OTC subunits aggregated. In contrast, when the chaperonin groEL was included in the dilution buffer, OTC did not aggregate but instead comigrated in a sucrose density gradient with the groEL oligomer, indicating that a complex had been formed. Upon addition of the cochaperonin groES and ATP to the isolated OTC-groEL complex, OTC monomers were folded, released, and assembled into active trimer. Neither groES nor ATP alone was sufficient to release active OTC from groEL. The extent of recovery of activity was proportional to the concentration of the complex, reaching approximately 80-90% at monomer concentrations above 0.6 microM. At low complex concentrations, kinetic studies revealed an initial lag in the reconstitution reaction, suggesting that assembly is the rate-limiting step under these conditions. We could trap folded, released, inactive OTC monomers at early times that assembled into active trimers with longer incubation. A nonhydrolyzable ATP analog could release bound OTC from groEL in the presence of groES, but the OTC monomers were not competent for assembly. These data show that recovery of OTC activity in vitro can be efficiently directed by the bacterial chaperonins in the presence of ATP and suggest that the mechanism of reconstitution involves ATP and groES-dependent folding and release of OTC monomers from groEL, followed by spontaneous assembly of trimers.

Adenosine Triphosphate↗
PubMed1979

Arrangement of proteins O-8 and O-9 in outer membrane of Escherichia coli K-12. Existence of homotrimers and heterotrimers.

1. The molecular arrangement of major outer membrane proteins O-8 and O-9 that exist as trimers has been studied by means of cross-linking with dimethylsuberimidate. 2. The cross-linked samples were examined on a urea/sodium dodecyl sulfate/polyacrylamide gel which was developed to separate cross-linked trimer and dimer of O-8 from those of O-9. 3. Cells simultaneously synthesizing both O-8 and O-9 formed heterotrimers (trimers containing both proteins) as well as homotrimers. 4. Quantitative analyses revealed that there was no discrimination between O-8 and O-9 in the assembly process to form trimers. 5. When cells were grown sequentially under two different sets of conditions so that the cells synthesized either one of the two proteins in the first stage and the other in the second stage of growth, no heterotrimers were formed. This result indicates that subunit exchange did not take place between trimers which had been incorporated into the outer membrane.

Cell Membrane↗
PubMed1995

Synthesis of chloramphenicol acetyltransferase in a coupled transcription-translation in vitro system lacking the chaperones DnaK and DnaJ.

A trimeric enzyme chloramphenicol acetyltransferase (CATI) has been synthesized in the Zubay system genetically depleted from DnaK and DnaJ. Most of CAT formed in the system fail to assemble into an active trimer. Instead CAT is accumulated in either a GroEL-bound complex or as an inactive monomer. Addition of purified DnaK and DnaJ to the system prior to the start of protein synthesis leads to the increase of the specific activity of formed CAT. A portion of exogenous DnaK and DnaJ added to the system associate with nascent polypeptide chains in the ribosomes. DnaK also comigrates with 50S-ribosomal subunits.

Bacterial Proteins↗
PubMed1996

Organization of 'nanocrystal molecules' using DNA.

Patterning matter on the nanometre scale is an important objective of current materials chemistry and physics. It is driven by both the need to further miniaturize electronic components and the fact that at the nanometre scale, materials properties are strongly size-dependent and thus can be tuned sensitively. In nanoscale crystals, quantum size effects and the large number of surface atoms influence the, chemical, electronic, magnetic and optical behaviour. 'Top-down' (for example, lithographic) methods for nanoscale manipulation reach only to the upper end of the nanometre regime; but whereas 'bottom-up' wet chemical techniques allow for the preparation of mono-disperse, defect-free crystallites just 1-10 nm in size, ways to control the structure of nanocrystal assemblies are scarce. Here we describe a strategy for the synthesis of 'nanocrystal molecules', in which discrete numbers of gold nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. We attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addition of a complementary single-stranded DNA template. We anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.

Base Sequence↗
PubMed1998

Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore.

The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C-terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore.

Adenosine Triphosphatases↗
PubMed1997

Assembly of a rod-shaped chimera of a trimeric GCN4 zipper and the HIV-1 gp41 ectodomain expressed in Escherichia coli.

The HIV-1 envelope subunit gp41 plays a role in viral entry by initiating fusion of the viral and cellular membranes. A chimeric molecule was constructed centered on the ectodomain of gp41 without the fusion peptide, with a trimeric isoleucine zipper derived from GCN4 (pIIGCN4) on the N terminus and part of the trimeric coiled coil of the influenza virus hemagglutinin (HA) HA2 on the C terminus. The chimera pII-41-HA was overexpressed as inclusion bodies in bacteria and refolded to soluble aggregates that became monodisperse after treatment with protease. Either trypsin or proteinase K, used previously to define a protease-resistant core of recombinant gp41 [Lu, M., Blacklow, S. C. & Kim, P. S. (1995) Nat. Struct. Biol. 2, 1075-1082], removed about 20-30 residues from the center of gp41 and all or most of the HA2 segment. Evidence is presented that the resulting soluble chimera, retaining the pIIGCN4 coiled coil at the N terminus, is an oligomeric highly alpha-helical rod about 130 A long that crystallizes. The chimeric molecule is recognized by the Fab fragments of mAbs specific for folded gp41. A similar chimera was assembled from the two halves of the molecule expressed separately in different bacteria and refolded together. Crystals from the smallest chimera diffract x-rays to 2.6-A resolution.

Antibodies↗
PubMed1997

Protein quality control along the route to the plant vacuole.

To acquire information on the relationships between structural maturation of proteins in the endoplasmic reticulum (ER) and their transport along the secretory pathway, we have analyzed the destiny of an assembly-defective form of the trimeric vacuolar storage glycoprotein phaseolin. In leaves of transgenic tobacco, where assembly-competent phaseolin is correctly targeted to the vacuole, defective phaseolin remains located in the ER or a closely related compartment where it represents a major ligand of the chaperone BiP. Defective phaseolin maintained susceptibility to endoglycosidase H and was slowly degraded by a process that is not inhibited by heat shock or brefeldin A, indicating that degradation does not involve transport along the secretory pathway. These results provide evidence for the presence of a quality control mechanism in the ER of plant cells that avoids intracellular trafficking of severely defective proteins and eventually leads to their degradation.

Biological Transport↗
PubMed2003

Fucoxanthin-chlorophyll proteins in diatoms: 18 and 19 kDa subunits assemble into different oligomeric states.

Fucoxanthin-chlorophyll proteins were purified from the centric diatom Cyclotella meneghiniana. Two major fractions were observed that differed in their polypeptide composition and oligomeric state. Trimers consist of mainly 18 kDa polypeptides. Higher oligomers are tightly assembled from different trimers, which contain mostly 19 kDa subunits. In both oligomeric states, the excitation energy coupling between fucoxanthin and chlorophyll a was preserved, and chlorophyll c was shown to transfer energy efficiently to chlorophyll a. Circular dichroism spectra showed close interaction between fucoxanthin and chlorophyll a, and different chlorophyll a molecules were demonstrated to interact excitonically. The assembly of trimers of antenna proteins with a distinct subunit composition into higher oligomeric states was not reported so far and differs from the situation found in higher plants. The differences in the supramolecular structure of the fucoxanthin-chlorophyll proteins reflect the dissimilar arrangement of the thylakoid membranes in diatoms, which lack the grana-stroma distinction.

Chromatography, Gel↗
PubMed2005

Assembly of the hexameric Escherichia coli arginine repressor investigated by nano-electrospray ionization time-of-flight mass spectrometry.

The arginine repressor (ArgR) from Escherichia coli regulates genes for L-arginine metabolism and is a required recombination factor for colE1 plasmid replication. Both functions require binding of L-arginine to the protein. In this work, nano-electrospray ionization time-of-flight mass spectrometry (nano-ESI-TOFMS) is used to study conformational and oligomeric states of intact ArgR and its isolated structural domains. In agreement with X-ray diffraction studies, it is shown that ArgR oligomerizes to form hexamers in both the presence and absence of L-arginine, and the basic unit of oligomerization appears to be the trimer. Higher-order assembly into dodecamers is also detected. The isolated C-terminal domain is found to associate into trimers and hexamers whereas the N-terminal domain is detected in its monomeric form. The observed species distributions suggest a role for the N-terminal domain in hexamer stabilization.

Escherichia coli↗
PubMed2003

Regulation of nicotinic acetylcholine receptor assembly.

The four muscle-type nicotinic acetylcholine receptor (AChR) subunits, alpha, beta, gamma, and delta, assemble into functional alpha(2)betagammadelta pentamers in the endoplasmic reticulum (ER) through a series of interdependent folding and oligomerization events. The first stable assembly intermediate is a trimer composed of alpha, beta, and gamma subunits. The formation of alphabetagamma trimers initiates a series of subunit folding and processing events that allow addition of delta subunits to form alphabetagammadelta tetramers. Subunit folding and processing continue with formation of the ligand-binding sites on the alpha subunit of alphabetagammadelta tetramers and the second alpha subunit added to assemble alpha(2)betagammadelta pentamers. AChR assembly is inefficient. Only 20-30% of synthesized subunits assemble into mature receptors in the ER, while the remaining unassembled subunits are degraded. However, the efficiency of subunit assembly can be regulated under certain conditions leading to higher AChR expression. Increased intracellular cAMP levels cause a 2- to 3-fold increase in AChR assembly efficiency and a comparable increase in surface expression. Additionally, block of ubiquitin-proteasome degradation appears to enhance AChR assembly and expression. Thus, the regulation of AChR assembly through posttranslational mechanisms is a potential therapeutic target for increasing AChR expression in diseases in which expression is compromised.

Animals↗
PubMed1995

Type X collagen multimer assembly in vitro is prevented by a Gly618 to Val mutation in the alpha 1(X) NC1 domain resulting in Schmid metaphyseal chondrodysplasia.

Type X collagen is a homotrimer of alpha 1(X) chains encoded by the COL10A1 gene. It is a highly specialized extracellular matrix component, and its synthesis is restricted to hypertrophic chondrocytes in the calcifying cartilage of the growth plate and in zones of secondary ossification. Our studies on a family with Schmid metaphyseal chondrodysplasia demonstrated that the affected individuals were heterozygous for a single base substitution in the COL10A1 gene, which changed the codon GGC for glycine 618 to GTC for valine in the highly conserved region of the carboxyl-terminal NC1 domain and altered the amino acid sequence in the putative oligosaccharide attachment site. Since hypertrophic cartilage tissues or cell cultures were not available to assess the effect of the mutation, an in vitro cDNA expression system was used to study normal and mutant type X collagen biosynthesis and assembly. Full-length cDNA constructs of the normal type X collagen sequence and also cDNA containing the specific Gly to Val NC1 mutation found in the patient were produced and expressed by in vitro transcription and translation. While the control construct produced type X collagen, which formed trimeric collagen monomers and assembled into larger multimeric assemblies, the mutant collagen was unable to form these larger aggregates. These experiments demonstrated that the mutation disturbed type X collagen NC1 domain interaction and assembly, a finding consistent with the abnormal disorganized cartilage growth plate seen in the patient. These studies provide the first evidence of the effect of a type X collagen mutation on protein structure and function and directly demonstrate the critical role of interactions between NC1 domains in the formation of type X collagen multimeric structures in vitro.

Amino Acid Sequence↗
PubMed2001

Hantavirus nucleocapsid protein oligomerization.

Hantaviruses are enveloped, negative-strand RNA viruses which can be lethal to humans, causing either a hemorrhagic fever with renal syndrome or a hantaviral pulmonary syndrome. The viral genomes consist of three RNA segments: the L segment encodes the viral polymerase, the M segment encodes the viral surface glycoproteins G1 and G2, and the S segment encodes the nucleocapsid (N) protein. The N protein is a 420- to 430-residue, 50-kDa protein which appears to direct hantavirus assembly, although mechanisms of N protein oligomerization, RNA encapsidation, budding, and release are poorly understood. We have undertaken a biochemical and genetic analysis of N protein oligomerization. Bacterially expressed N proteins were found by gradient fractionation to associate not only as large multimers or aggregates but also as dimers or trimers. Chemical cross-linking of hantavirus particles yielded N protein cross-link products with molecular masses of 140 to 150 kDa, consistent with the size of an N trimer. We also employed a genetic, yeast two-hybrid method for monitoring N protein interactions. Analyses showed that the C-terminal half of the N protein plus the N-terminal 40 residues permitted association with a full-length N protein fusion. These N-terminal 40 residues of seven different hantavirus strains were predicted to form trimeric coiled coils. Our results suggest that coiled-coil motifs contribute to N protein trimerization and that nucleocapsid protein trimers are hantavirus particle assembly intermediates.

Amino Acid Motifs↗
PubMed2001

Interaction between molecules of hantavirus nucleocapsid protein.

Intermolecular interactions of Tula hantavirus N (nucleocapsid) protein were detected in the yeast two-hybrid system, prompting further attempts to study this phenomenon. Using chemical cross-linking and immunoblotting it was shown that the N protein from purified virus and from infected cell lysates as well as recombinant protein produced in a baculovirus expression system are capable of forming dimers, trimers and multimers, thus confirming the capacity of the protein molecules to interact with each other. An ELISA format was developed in which molecules of the recombinant N protein were shown to associate non-covalently, via electrostatic interactions. Divalent cations (Ca(2+), Mn(2+), Mg(2+), Ba(2+)) enhanced the process 3- to 8-fold suggesting that adequate folding of the N protein is crucial for the association. Based on these data a model for hantavirus nucleocapsid assembly is proposed, in which N molecules first trimerize around the viral RNA molecule, and then the trimers gradually assemble forming longer multimers.

Animals↗
PubMed1998

Formation of the nicotinic acetylcholine receptor binding sites.

Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different alpha subunits. The two alpha subunits, alpha gamma and alpha delta, are distinguished by their interface with gamma and delta subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on alpha gamma subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on alpha beta gamma delta subunit tetramers soon after the site for the competitive antagonist alpha-bungarotoxin has formed and delta subunits have assembled with alpha beta gamma trimers. The second site is located on alpha delta subunits and forms after AChR subunits have assembled into alpha2 beta gamma delta pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the delta and second alpha subunits associate during subunit assembly.

Animals↗
PubMed1995

In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity.

The coat protein that forms the icosahedral shell of phage P22 can be efficiently refolded in vitro [Teschke, C. M., & King, J. (1993) Biochemistry 32, 10839-10847]. Temperature-sensitive mutants of coat protein interfere with folding or assembly in vivo [Gordon, C. L., & King, J. (1993) J. Biol. Chem. 268, 9358-9368]. The folding of a set of phage P22 coat proteins carrying the temperature-sensitive for folding (tsf) substitutions W48Q, A108V, G232D, T294I, and F353L has been investigated in vitro. Denatured tsf polypeptides were able to fold into soluble species with high efficiency. The efficiency of folding of the wild-type (WT) and mutant polypeptides at different temperatures showed sharp transitions where aggregation predominated over folding. The refolding of the tsf mutant proteins did not show an obvious thermal defect in yield. The tsf polypeptides folded through the long-lived kinetic intermediate previously described for WT coat protein with similar relaxation times. The folding kinetics of the tsf polypeptides in bisANS, a hydrophobic fluorescent dye, were also similar to those of the WT protein. However, the folded tsf proteins showed decreased secondary structure compared to WT coat protein. Analysis of the folded state by native gel electrophoresis revealed that the tsf coat proteins folded into dimers and trimers, not monomers. The dimer and trimer species were incompetent for assembly. Once formed, dimers and trimers showed no propensity toward aggregation. The folding pathway of the mutant polypeptides must be quite similar to the WT pathway, but at some step inappropriate intersubunit interactions occur due to the amino acid substitutions, trapping the subunits from assembly.

Anilino Naphthalenesulfonates↗
PubMed2000

Trp203 mutation in GroEL promotes a self-association reaction: a hydrodynamic study.

A combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge is used to investigate the hydrodynamic integrity and increased self-association interactions of the mutant GroEL Y203W when compared to the wild-type GroEL molecule, which may be derived from increased hydrophobic exposure caused by the mutation. Sedimentation velocity has revealed that three distinct species were present throughout the concentration ranges used, corresponding to 14-mer (GroEL "super monomer") and 28-mer ("super dimer") subunit compositions with a small amount of 42-mer ("super trimer"), which, from the relative concentration of each species, would give an estimated weight average molecular weight of (1.0 +/- 0.1) x 10(6) Da. Sedimentation equilibrium gave an apparent weight average molecular weight (Mw,app) of (910,000 +/- 5000) Da, which is in agreement with these findings. These results are in contrast to wild-type GroEL which, in excellent agreement with the previous findings of Behlke and co-workers, revealed a single species with an Mw,app of (805,000 +/- 5200) Da and a sedimentation coefficient s(0)20,w of (21.6 +/- 0.3) S. We therefore conclude that the tryptophan mutation at the Y203 location causes a significant degree of self-association of the GroEL 14-mer assembly (with dimer and trimer present). These findings would appear to correlate well with the findings of Gibbons et al., who showed an increase in hydrophobic exposure due to this mutation.

Amino Acid Substitution↗
PubMed2006

Assembly of multimeric phage nanostructures through leucine zipper interactions.

One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.

Bacteriophage M13↗
PubMed1998

Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein sigma1.

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

Adenosine Triphosphate↗