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.     

Effect of structural modifications on the assembly of a glycinin subunit.

A Gy4 glycinin cDNA was modified and used to produce structurally altered 11S storage protein subunits. We evaluated these modified subunits for their ability to assemble into oligomers. Alterations made in the acidic polypeptide changed the subunit solubility characteristics but did not eliminate assembly. Modifications in the basic polypeptide usually eliminated assembly of subunits into trimers. A region exhibiting high natural variability located at the COOH terminus of the acidic polypeptide that we have designated the hypervariable region was also studied. Extensive deletions and insertions were tolerated in the hypervariable region without perturbing subunit assembly. Some of the insertions significantly increased the methionine content in the Gy4 glycinin subunit. Together, our results indicated that the structure of the basic polypeptide was more critical for assembly of trimers than that of the acidic polypeptide, an observation that implies that the basic polypeptides direct trimer formation. The assembly assays described here will be useful in efforts to improve seed quality. Using them, the effects of modifications to the storage protein subunits can be rapidly evaluated before introducing the mutated genes into plants.     

P22 tailspike trimer assembly is governed by interchain redox associations

Though disulfide bonds are absent from P22 tailspike protein in its native state, a disulfide-bonded trimeric intermediate has been identified in the tailspike folding and assembly pathway in vitro. The formation of disulfide bonds is critical to efficient assembly of native trimers as mutations at C-terminal cysteines reduce or inhibit trimer formation. We investigated the effect of different redox folding environments on tailspike formation to discover if simple changes in reducing potential would facilitate trimer formation. Expression of tailspike in trxB cell lines with more oxidizing cytoplasms led to lower trimer yields; however, observed assembly rates were unchanged. In vitro, the presence of any redox buffer decreased the overall yield compared to non-redox buffered controls; however, the greatest yields of the native trimer were obtained in reducing rather than oxidizing environments at pH 7. Slightly faster trimer formation rates were observed in the redox samples at pH 7, perhaps by accelerating the reduction of the disulfide-bonded protrimer to the native trimer. These rates and the effects of the redox system were found to depend greatly on the pH of the refolding reaction. Oxidized glutathione (GSSG) trapped a tailspike intermediate, likely as a mixed disulfide. This trapped intermediate was able to form native trimer upon addition of dithiothreitol (DTT), indicating that the trapped intermediate is on the assembly pathway, rather than the aggregation pathway. Thus, the presence of redox agents interfered with the ability of the tailspike monomers to associate, demonstrating that disulfide associations play an important role during the assembly of this cytoplasmic protein.     

In vitro oligomerization of a membrane protein complex. liposome-based reconstitution of trimeric photosystem I from isolated monomers.

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacterium Spirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl beta-D-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA-PsaF and PsaI-PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.     

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular determinants of self-association and rearrangement of a trimeric intermediate during the assembly of a parvovirus capsid

The minute virus of mice (MVM) provides a simple model for the dissection of the molecular determinants of the self-assembly, stability, and dynamics of a biological supramolecular complex. MVM assembly involves the trimerization of capsid subunits in the cytoplasm; trimers are transported to the nucleus, where they suffer a conformational change and are made competent for capsid formation. Our previous study revealed that capsid assembly from trimers is dependent on stronger intertrimer interactions that are equally spaced in an equatorial belt surrounding each trimer. We have now targeted the interfaces between monomers within each trimer to identify the molecular determinants of trimerization and the rearrangement needed for capsid assembly. Twenty-eight amino acid residues per monomer were individually mutated to alanine to remove most of the stronger intersubunit interactions. The effects on trimer and capsid assembly and virus infectivity in cells were analyzed. No side chain was individually required for trimer assembly in the cytoplasm; in contrast, half of them were required to make the trimers competent for nuclear capsid assembly, even though none was close to intertrimer interfaces. These critical side chains are conserved and participate in extensive hydrophobic contacts, buried hydrogen bonds, or salt bridges between subunits. This study on MVM capsid assembly reveals that: (i) trimerization is a robust process, insensitive to removal of individual intersubunit interactions; and (ii) the rearrangement of the trimer intermediate required for capsid assembly is a global process that depends on the establishment of many interactions along the protein-protein interfaces within each trimer.     

Allophycocyanin: trimers,monomers, subunits,and homodimers

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.     

Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli

Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.     

Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12.

Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants.     

Assembly pathway of newly synthesized LamB protein an outer membrane protein of Escherichia coli K-12

The assembly of newly induced LamB protein (phage lambda receptor) was investigated in an operon fusion strain of Escherichia coli, in which the lamB gene is expressed under lac promoter control. The induction kinetics both for total cellular and for cell surface-exposed LamB protein were studied by immunochemical detection methods, using two distinct antisera directed against detergent-solubilized LamB trimers and completely denatured LamB monomers, respectively. Anti-trimer antibodies recognized both monomers and trimers, whereas anti-monomer antibodies only reacted with monomers. Provided appropriate solubilization conditions were used, both antisera were able to immunoprecipitate intracellular mature LamB protein quantitatively. Following induction, the first LamB antigenic determinants were detected after 60 to 80 seconds; detection of the newly synthesized protein by anti-monomer antibodies slightly preceded that by anti-trimer antibodies, a finding that could be partly explained by the observation that anti-monomer antibodies recognized a larger fraction of nascent LamB than did anti-trimer antibodies. Exposure of antigenic determinants at the cell surface was delayed for 30 to 50 seconds with respect to their synthesis. Therefore, either translocation or conformational changes must be rate-limiting in the series of processes that eventually convert the newly synthesized protein into its mature outer membrane state. LamB protein was found to occur in at least three clearly distinguishable states. State I is the LamB monomer, state II corresponds to a metastable trimer that dissociates in sodium dodecyl sulphate above 60 degrees C, and state III is the state LamB trimer that dissociates in sodium dodecyl sulphate only at temperatures above 90 degrees C. The chase kinetics of these states showed that conversion of newly synthesized LamB monomers to stable LamB trimers occurred in two stages: state I monomers were chased into metastable state II trimers rapidly (t 1/2 = 20 s), whereas stabilization of state II trimers to state III trimers was a relatively slow (t 1/2 = 5.7 min) process. Based on our results, a timing sequence in the assembly of outer membrane LamB protein is proposed.     

Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-kappaB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-alpha at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.     

Mutational analysis of N381, a key trimer contact residue in Tsr, the Escherichia coli serine chemoreceptor

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.     

Synthesis and processing of a type I procollagen containing shortened pro-alpha 1(I) chains by fibroblasts from a patient with osteogenesis imperfecta

Skin fibroblasts from a patient with a lethal form of osteogenesis imprefecta were found to synthesize equal amounts of normal pro-alpha 1(I) chains and pro-alpha 1(I) chains which are about 10% shorter because of a deletion of about 100 amino acids in the middle of the alpha chain domain. The pro-alpha 1(I) chains were incorporated into three different kinds of trimers: a normal type I trimer with normal length pro-alpha 1(I) chains; a type Is trimer with one shortened pro-alpha 1(I) chain and two normal length chains; and a type Iss trimer containing two shortened pro-alpha 1(I) chains and one normal length pro-alpha 2(I) chain. As judged by resistance to digestion by chymotrypsin and trypsin, the type Is and Iss trimers denatured at a temperature at least 3 degrees C lower than normal type I procollagen. Procollagen containing the shortened pro-alpha 1(I) chains was slowly secreted by the cells but was degraded by extracellular proteinases within 6 h of chase into the medium. The results indicated that the presence of the shortened pro-alpha 1(I) chains in procollagen trimers produces a delay in rate of helix formation, overmodification of the polypeptides by post-translational enzymes, a decrease in the thermal stability of the trimers, and increased susceptibility of the protein to endogenous proteinases. Additionally, the fibroblasts of this patient synthesized and secreted a type III-like species of procollagen with unusual chromatographic properties.     

Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers

We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.     

Structure/function relationships in the minicollagen of Hydra nematocysts

The minicollagens found in the inner layer of the Hydra nematocyst walls are the smallest collagens known with 12-16 Gly-X-Y repeats. Minicollagen-1, the best characterized member of this protein family so far, consists of a central collagen triple helix of 12 nm in length flanked at both ends by a polyproline stretch and a conserved cysteine-rich domain. The cysteine-rich tails are proposed to function in the assembly of soluble minicollagen trimers to high molecular structures by a switch of the disulfide linkage from intramolecular to intermolecular bonds. In this study, we investigate the trimeric nature of minicollagen-1 and its capacity to form disulfide-linked polymers in vitro. A fusion protein of minicollagen-1 with maltose-binding protein is secreted as a soluble trimer with only intrachain and no interchain disulfide bridges as confirmed by melting the collagen triple helix under reducing and non-reducing conditions. The conversion of minicollagen-1 trimers to monomers takes place between 40 and 55 degrees C with the melting point being approximately 45 degrees C. Oxidative reshuffling of the minicollagen-1 trimers leads to the formation of high molecular aggregates, which upon reduction show distinct polytrimeric states. Minicollagen trimers in isolated nematocyst capsules proved to be sensitive to SDS and were engaged in polymeric structures with additional cross-links that were resistant to reducing agent.     

Role of posttranslational cleavage in glycinin assembly.

Glycinin, like other 11S seed storage proteins, undergoes a complex series of posttranslational events between the time proglycinin precursors are synthesized in endoplasmic reticulum and the mature glycinin subunits are deposited in vacuolar protein bodies. According to the current understanding of this process, proglycinin subunits aggregate into trimers in endoplasmic reticulum, and then the trimers move to the vacuolar protein bodies where a protease cleaves them into acidic and basic polypeptide chains. Stable glycinin hexamers, rather than trimers, are isolated from mature seeds. We used a re-assembly assay in this study to demonstrate that proteolytic cleavage of the proglycinin subunits is required for in vitro assembly of glycinin oligomers beyond the trimer stage. The possibility that the cleavage is a regulatory step and that it triggers the deposition of 11S seed storage proteins as insoluble aggregates in vivo is considered.