Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Trimer assembly”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 595 records · Page 33Linked to original sources
PubMed1994

Pyruvate dehydrogenase multienzyme complex. Characterization of assembly intermediates by sedimentation velocity analysis.

The pyruvate dehydrogenase complex is a large, highly organized assembly of several different catalytic and regulatory component enzymes. The structural core of the complex is the E2-X subcomplex, consisting of 60 dihydrolipoamide transacetylase (E2) subunits arranged in a pentagonal dodecahedron; 6 protein X and 2 pyruvate dehydrogenase kinase molecules are tightly associated with this E2 60-mer. The native E2-X subcomplex exhibits a sedimentation coefficient of 32 S. The effects of several chaotropes (guanidinium chloride, potassium thiocyanide, and urea) on the E2-X subcomplex were assessed. Treatment of the E2-X subcomplex with 4 M guanidinium chloride caused a complete loss of enzymatic activity and the dissociation of the subcomplex into monomeric 1.5-3 S species. Removal of the chaotrope by dialysis for 18 h resulted in complete restoration of E2 enzymatic activity and reassembly of a 32 S subcomplex; this reassembled subcomplex contained less protein X than the native subcomplex. Sedimentation velocity analysis of reassembled E2-X subcomplex demonstrated the presence of an 8 S assembly intermediate; this sedimentation coefficient is characteristic of globular proteins of molecular weights similar to that expected for a trimer of E2. Shorter periods of dialysis also gave rise to the 8 S species; the amount of this intermediate decreased with increasing times of dialysis. The 8 S species associated non-cooperatively to yield additional assembly intermediates exhibiting sedimentation coefficients of 10-32 S.

Acetyltransferases↗
PubMed2001

Oligonucleotide-directed assembly of materials: defined oligomers.

A nano-architectural system that has high variability while maintaining component specificity is described. Tetraphenylcyclobutadiene(cyclopentadienyl)cobalt complexes and phenyleneethynylene trimers were synthesized and subsequently modified with oligonucleotides utilizing standard phosphoramidite chemistry. The resulting oligonucleotide modified organics (OMOs) were characterized by UV-vis spectroscopy, fluorescence spectroscopy, and phosphate analysis. Hybridization of these OMOs resulted in a series of self-assembled oligomeric hybrids of varying length and topology. These hybrids were characterized by melting temperature, polyacrylamide gel electrophoresis, and fluorescence spectroscopy. This model system demonstrates the power of DNA to self-assemble modules of interest-independent of the module itself.

DNA↗
PubMed2002

Duplex oligomers defined via covalent casting of a one-dimensional hydrogen-bonding motif.

Hydrogen-bonded tapes comprised of monomeric molecular precursors are used to define structural parameters for the design of related oligomers encoded with predetermined modes of assembly. Application of this "covalent casting" strategy vis-à-vis the one-dimensional H-bonding motif expressed by 2-amino-4,6-dichlorotriazine has enabled the design of high-affinity duplex molecular strands. Dimeric, trimeric, and tetrameric duplex oligomers are prepared through an iterative synthetic protocol involving sequential homologation of the oligo(aminotriazine). The mode of assembly and interstrand affinity of homologous oligomers are established in solution by (1)H NMR dilution experiments, isothermal titration calorimetry (ITC), vapor pressure osmometry (VPO), cross-hybridization experiments involving the analysis of dye-labeled strands via thin-layer chromatography (TLC), and in the solid state by X-ray crystallographic analysis. Binding free energy per unimer (-Delta G degrees/n) increases significantly upon extension from monomer to dimer to trimer, signifying a strong positive cooperative effect. Nanomolar binding affinity (K(d) = 1.44 +/- 0.50 nM) was determined for the duplex trimer by ITC in 1,2-dichloroethane at 20 degrees C. In-register duplex formation is not observed for the tetramer, which appears to adopt an alternative binding mode. These data give insight into the structural and interactional features of the oligomers required for high-affinity, high-specificity binding and define a platform for the design of second-generation systems and related duplex strands for use in nanoscale assembly.

Calorimetry↗
PubMed1990

Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides.

The associations of both rough and smooth lipopolysaccharides (LPS) with the OmpF porin of Escherichia coli K-12 were examined in galE strains deleted for ompC. Transformation with pSS37 and growth with galactose conferred the ability to assemble a Shigella dysenteriae O antigen onto the core oligosaccharide of E. coli K-12 LPS. The association of LPS with OmpF trimers was assessed by staining, autoradiography of LPS specifically labeled with [1-14C]galactose, and Western immunoblotting with a monoclonal antibody specific for OmpF trimers. These techniques revealed that the migration distances and multiple banding patterns of OmpF porin trimers in sodium dodecyl sulfate-polyacrylamide gels were dictated by the chemotype of associated LPS. Expression of smooth LPS caused almost all of the trimeric OmpF to run in gels with a slower mobility than trimers from rough strains. The LPS associated with trimers from a smooth strain differed from the bulk-phase LPS by consisting almost exclusively of molecules with O antigen.

Autoradiography↗
PubMed2005

Weak hydrogen bonds as a structural motif for two-dimensional assemblies of oligopyridines on highly oriented pyrolytic graphite: an STM investigation.

We present the STM investigation of four different oligopyridines at the liquid/highly oriented pyrolytic graphite interface. The heteroaromatic compounds are constitutional isomers showing the same overall shape regardless of their actual conformation. On the basis of weak intermolecular C-H...N hydrogen-bonding interactions, different nanopatterns are formed following a simple general concept for the two dimensional self-assembly. The molecules arrange either in linear or in cyclic structures. Though the oligopyridines are achiral, the formation of prochiral trimeric superstructures leads to chiral phases due to the immobilization on the surface. Some of the molecules show polymorphic structures depending on the solvent. The large variety of the presented structures formed by self-assembly of the different oligopyridines which retain the same functional heteroaromatic backbone shall open the possibility of exploiting these patterns as templates for the nanostructuring of surfaces with guests such as small molecules or metal ions for intriguing applications in, for example, catalysis.

Journal Article↗
PubMed2005

On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers.

Annexin A5 is a protein that binds to membranes containing negatively charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein's 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with atomic force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concentration of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concentration in solution. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (< or =10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concentration. The adsorption is determined by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.

Adsorption↗
PubMed1995

Characterization of class I MHC folding intermediates and their disparate interactions with peptide and beta 2-microglobulin.

Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (beta 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2Ld and Lq, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of Ld or Lq, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with beta 2m and these L alt/beta 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain/beta 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/beta 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with beta 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the alpha 2 domain disulfide bond, accumulated intracellularly, were not beta 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of Ld alt to conformed Ld. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with beta 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.

Animals↗
PubMed2004

Insights into assembly from structural analysis of bacteriophage PRD1.

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

Amino Acid Sequence↗
PubMed2006

Biosynthesis and enzymology of the Caenorhabditis elegans cuticle: identification and characterization of a novel serine protease inhibitor.

Caenorhabditis elegans represents an excellent model in which to dissect the biosynthesis and assembly of the nematode cuticle. A sequenced genome, straightforward transgenesis, available mutants and practical genome-wide RNAi approaches provide an invaluable toolkit in the characterization of cuticle components. We have performed a targeted RNAi screen in an attempt to identify components of the cuticle collagen biosynthetic pathway. Collagen biosynthesis and cuticle assembly are multi-step processes that involve numerous key enzymes involved in post-translational modification, trimer folding, procollagen processing and subsequent cross-linking stages. For many of these steps, the modifications and the enzymes are unique to nematodes and may represent attractive targets for the control of parasitic nematodes. A novel serine protease inhibitor was uncovered during our targeted screen, which is involved in collagen maturation, proper cuticle assembly and the moulting process. We have confirmed a link between this inhibitor and the previously uncharacterised bli-5 locus in C. elegans. The mutant phenotype, spatial expression pattern and the over-expression phenotype of the BLI-5 protease inhibitor and their relevance to collagen biosynthesis are discussed.

Animals↗
PubMed2002

Assembly of the RAG1/RAG2 synaptic complex.

Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.

DNA↗
PubMed2006

Amyloid beta-peptide oligomerization in silico: dimer and trimer.

Soluble oligomers of Alzheimer's amyloid beta protein (Abeta) may act as effectors of neurotoxicity in early stages of Alzheimer's disease. Detailed information about the structure of Abeta in atomistic level and the dynamics of assembly of monomeric Abeta into oligomeric structures is rather elusive. We have performed replica exchange molecular dynamics (REMD) simulations on the formation of the dimer and trimer of Abeta10-35 peptide. We have observed spontaneous formation of several basic structural units that may act as a template or an intermediate for further aggregation of Alzheimer's Abeta protein. Various conformers, including interlocking structures of experimentally known bend double beta strands, are identified.

Alzheimer Disease↗
PubMedDate not supplied

Channel properties of template assembled alamethicin tetramers.

The multiple conductance levels displayed by the antibiotic alamethicin in planar lipid bilayers is explained by a dynamic 'barrel-stave' model, the conducting pore resulting from the aggregation of up to ten helical amphipathic helical monomers. However, the precise assignment of an oligomerization state to a particular single-channel conductance substate is far from being experimentally clear. In addition, it could be useful to tailor a given channel geometry to selectively allow the permeation of solutes with different molecular sizes, whilst retaining a high voltage-dependence. To control the aggregation state of the channel, the TASP (template assembled synthetic proteins) strategy was applied to synthesize structurally defined oligomers, i.e. dimer, trimer, tetramer. The modulation of conductance properties of three alamethicin tetramers with the length and flexibility of the linkers of the 'open' or linear template is described. It is shown that the introduction of an alanine between the contiguous lysines to which are tethered C-terminally modified alamethicin helical monomers stabilizes the open channel states, whereas the alanine substitution by Pro-Gly, a reverse beta-turn promoting motif, increases voltage-dependence and leads to single-channel conductance values more in line with the expected ones from a tetrameric bundle.

Alamethicin↗
PubMed1997

Deletion mutants of the herpes simplex virus type 1 UL8 protein: effect on DNA synthesis and ability to interact with and influence the intracellular localization of the UL5 and UL52 proteins.

The herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric complex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 protein has been implicated in modulating these functions, facilitating efficient nuclear uptake of the complex and interacting with other viral DNA replication proteins. To further our understanding of UL8, we have constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolished the ability of UL8 to support DNA replication in transient transfection assays. None of the UL8 mutants tested exhibited a strong dominant negative phenotype in the presence of the wild-type product, although some inhibition of replication was observed with mutants lacking 165 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mutants to facilitate efficient nuclear localization of UL52 in the presence of coexpressed UL5 was examined by immunofluorescence. Selected mutants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The replicative ability of the mutants was found to correlate with their ability to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification of any region of UL8 important for its presumed nuclear functions.

Animals↗
PubMed2005

A survey of flexible protein binding mechanisms and their transition states using native topology based energy landscapes.

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interactions. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. Phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated Phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The Phi values for the formation of an antibody-antigen complex indicate a possible role for solvation of the interface in biomolecular association of large rigid proteins.

Antigen-Antibody Complex↗
PubMed1995

The Binding Protein Associates with Monomeric Phaseolin.

The association of the binding protein (BiP) with newly synthesized proteins in the endoplasmic reticulum (ER) of developing bean (Phaseolus vulgaris) cotyledonary cells was investigated. ATP-sensitive association with many polypeptides was detected. The fraction of newly synthesized polypeptides associated with BiP varies among different proteins. The relationship between subunit assembly and binding to BiP was investigated in the case of the vacuolar trimeric glycoprotein phaseolin. In spite of the presence of a significant pool of phaseolin trimers in the ER, only monomeric phaseolin is found in association with BiP. On the whole, our results point to a general role of BiP in the synthesis of plant secretory proteins and indicate that, in the case of phaseolin, BiP binding sites are concealed during structural maturation in the ER, either before or upon formation of trimers. Our results also indicate that trimerization does not constitute a rate-limiting step in the transport of phaseolin to the protein storage vacuoles.

Journal Article↗
PubMed2006

Surfactant protein A--from genes to human lung diseases.

Surfactant associated protein-A (SP-A) is the most abundant pulmonary surfactant protein and belongs to the family of innate host defense proteins termed collectins. Besides pulmonary host defense, SP-A is also involved in the formation of pulmonary surfactant, as it is essential for the structure of tubular myelin. The human SP-A gene locus includes two functional genes, SFTPA1 and SFTPA2 which are expressed independently, and a pseudo gene. The largest amount of SP-A1 proteins assemble to larger molecular complexes, whereas SP-A2 forms mainly dimers and trimers. SP-A polymorphisms play a role in respiratory distress syndrome, allergic bronchopulmonary aspergillosis and idiopathic pulmonary fibrosis. The levels of SP-A are decreased in the lungs of patients with cystic fibrosis, respiratory distress syndrome and further chronic lung diseases. Future areas for clinical research include disease specific SP-A expression pattern and their functional consequences, the differential roles of SP-A1 and SP-A2 in human lung diseases, and therapeutical approaches to correct altered SP-A levels.

Humans↗
PubMed1995

The phage T4-coded DNA replication helicase (gp41) forms a hexamer upon activation by nucleoside triphosphate.

Sedimentation and high performance liquid chromatography studies show that the functional DNA replication helicase of bacteriophage T4 (gp41) exists primarily as a dimer at physiological protein concentrations, assembling from gp41 monomers with an association constant of approximately 10(6) M-1. Cryoelectron microscopy, analytical ultracentrifugation, and protein-protein cross-linking studies demonstrate that the binding of ATP or GTP drives the assembly of these dimers into monodisperse hexameric complexes, which redissociate following depletion of the purine nucleotide triphosphatase (PuTP) substrates by the DNA-stimulated PuTPase activity of the helicase. The hexameric state of gp41 can be stabilized for detailed study by the addition of the nonhydrolyzable PuTP analogs ATP gamma S and GTP gamma S and is not significantly affected by the presence of ADP, GDP, or single-stranded or forked DNA template constructs, although some structural details of the hexameric complex may be altered by DNA binding. Our results also indicate that the active gp41 helicase exists as a hexagonal trimer of asymmetric dimers, and that the hexamer is probably characterized by D3 symmetry. The assembly pathway of the gp41 helicase has been analyzed, and its structure and properties compared with those of other helicases involved in a variety of cellular processes. Functional implications of such structural organization are also considered.

Adenosine Triphosphate↗
PubMedDate not supplied

A switch in disulfide linkage during minicollagen assembly in hydra nematocysts or how to assemble a 150-bar-resistant structure.

Hydra minicollagen, the shortest collagen known, is an important component of the nematocyst wall, which has a very high tensile strength. It has an unusual structure, with small and closely related Cys-rich domains at both ends of its chains. Three chains are trimerized by a central collagenous domain. Polyhydroxyproline helices connect the Cys-rich domains with the collagenous domain. The minicollagen precursor contains three internal disulfide bridges in each Cys-rich domain and no disulfide bridges between chains of the same trimeric molecule or between different molecules. Biochemical and structural evidence as well as confocal immunofluorescence microscopy points to disulfide-mediated assembly during maturation of nematocysts.

Animals↗