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PubMed1999

Engineered assembly of intertwined oligomers of an immunoglobulin chain.

Domain 1 of CD2 (CD2.D1) forms a conventional Ig fold stabilised by non-covalent antiparallel contacts between beta-strands. Removing two residues from the middle of the protein sequence, where the polypeptide chain normally folds back upon itself, stabilises an open conformation. In this modified molecule, the optimum evolved contacts between side-chains can only be satisfied through the antiparallel association of two chains to create a symmetrical pair of pseudo-domains. Here, we describe the dynamics of the switch between monomeric and dimeric states and demonstrate the extension of this novel underlying principle to trimer and tetramer formation. The ability of a protein molecule to form higher-order antiparallel structures is reminiscent of the behaviour of hairpins, duplexes, three-way and Holliday junctions in DNA.

Animals↗
PubMed1993

The unusual light-harvesting complex of Mantoniella squamata: supramolecular composition and assembly.

The supramolecular properties of the chlorophyll a/b/c light-harvesting protein complex of Mantoniella squamata were analyzed. The complex is built up of at least two subunits of M(r) 20,000 and 22,000, which are encoded in the nucleus as precursor proteins of M(r) 27,000. The chlorophyll a/b/c light-harvesting complex is the dominating protein of the thylakoids and is fractured with the protoplasmic membrane face as a 7.5 nm particle. These particles form paracrystalline arrays with a purported hexagonal arrangement in native thylakoids and form similar arrays when reconstituted in liposomes. The light-harvesting complexes are supposed to be trimers with a trigonal arrangement of the subunits. Preliminary amino acid sequence data show that the chlorophyll a/b/c light-harvesting complex of M. squamata is more related to the chlorophyll a/b complex of higher plants than to the light-harvesting complexes of chromophytan algae.

Amino Acid Sequence↗
PubMed2005

Collaborative signaling by bacterial chemoreceptors.

Motile bacteria seek optimal living habitats by following gradients of attractant and repellent chemicals in their environment. The signaling machinery for these chemotactic behaviors, although assembled from just a few protein components, has extraordinary information-processing capabilities. Escherichia coli, the best-studied model, employs a networked cluster of transmembrane receptors to detect minute chemical stimuli, to integrate multiple and conflicting inputs, and to generate an amplified output signal that controls the cell's flagellar motors. Signal gain arises through cooperative action of chemoreceptors of different types. The signaling-teams within a receptor cluster may be built from trimers of receptor dimers that communicate through shared connections to their partner signaling proteins.

Chemotaxis↗
PubMed2001

A novel dinuclear ruthenium complex bridged through a substituted phenazine ligand formed by ruthenium-promoted oxidative assembly of 1,3-diaminobenzene.

The reaction of [Ru(acac)3] (acac = acetylacetonate) with molten 1,3-diaminobenzene affords the crystalline monometallic compound [Ru(L1)-(acac)21 (1: L1 = N-(3'-aminophenyl)1,2-(3-amino)benzoquinone diimine) along with an unstable dimetallic compound [Ru2(mu-L2)(acac)4] (2: L2=N-4,6-bis(3'-aminophenyl)imino-3,5-diimino-hex-1-ene). Compound 2 transforms to a stable dimetallic compound [Ru2(mu-L3)(acac)4] (3: L3 = 2-amino-6(3'-aminophenyl)imino-9-imino-phenazine) in boiling 2-methoxyethanol. The above compounds are formed by ruthenium-mediated oxidative di- or trimerization of the diamine with the formation of several new C-N bonds. The products have been thoroughly characterized. FAB mass spectra, along with other physicochemical data, were used for their formulations. The compounds 1, 2, and 3 display intense peaks due to their parent molecular ions at m/z 512, 916, and 914, respectively. Final characterization of complex 3 was made by single-crystal X-ray structure determination. The structure of 3 confirmed the formation of three new C-N bonds and the bridging ligand L3 from 1,3-diaminobenzene. The conversion, 2 --> 3 is an oxidative ring-closure reaction, which is associated with dehydrogenation reactions. The monometallic compound 1, showed a reversible metal-based anodic response at 0.35 V. On the other hand, both the compounds 2 and 3 showed a pair of well-resolved metal-based anodic oxidations, for which the separation between the two successive anodic responses were high (>0.4 V). In addition, all of them showed multiple cathodic responses that were in the range -1.0 to -2.0 V.

Journal Article↗
PubMed2005

[MnIII2 (5-Rsaltmen)2NiII(pao)2(L)]2+: an S(T)=3 building block for a single-chain magnet that behaves as a single-molecule magnet.

Mn(III)-Ni(II)-Mn(III) linear-type trinuclear complexes bridged by oximate groups were selectively synthesized by the assembly reaction of [Mn2(5-Rsaltmen)2(H2O)2](ClO4)2 (5-Rsaltmen2-=N,N'-(1,1,2,2-tetramethylethylene) bis(5-R-salicylideneiminate); R=Cl, Br) with [Ni(pao)2(phen)] (pao-=pyridine-2-aldoximate; phen=1,10-phenanthroline) in methanol/water: [Mn2(5-Rsaltmen)2Ni(pao)2(phen)](ClO4)2 (R=Cl, 1; R=Br, 2). Structural analysis revealed that the [Mn(III)-ON-Ni(II)-NO-Mn(III)] skeleton of these trimers is in every respect similar to the repeating unit found in the previously reported series of 1D materials [Mn2(saltmen)2Ni(pao)2(L1)2](A)(2) (L(1)=pyridine, 4-picoline, 4-tert-butylpyridine, N-methylimidazole; A=ClO4-, BF4-, PF6-, ReO4-). Recently, these 1D compounds have attracted a great deal of attention for their magnetic properties, since they exhibit slow relaxation of the magnetization (also called single-chain magnet (SCM) behavior). This unique magnetic behavior was explained in the framework of Glauber's theory, generalized for chains of ferromagnetically coupled anisotropic spins. Thus, in these 1D compounds, the [Mn(III)-ON-Ni(II)-NO-Mn(III)] unit was considered as an S(T)=3 anisotropic spin. Direct-current magnetic measurements on 1 and 2 confirm their S(T)=3 ground state and strong uniaxial anisotropy (D/k(B) approximately -2.4 K), in excellent agreement with the magnetic characteristic deduced in the study on the SCM series. The ac magnetic susceptibility of these trimers is strongly frequency-dependent and characteristic of single-molecule magnet (SMM) behavior. The relaxation time tau shows a thermally activated (Arrhenius) behavior with tau0 approximately 1x10(-7) s and Delta(eff)/k(B) approximately 18 K. The effective energy barrier for reversal of the magnetization Delta(eff) is consistent with the theoretical value (21 K) estimated from |D| S2T. The present results reinforce consistently the interpretation of the SCM behavior observed in the [Mn2(saltmen)2Ni(pao)2(L1)2](A)2 series and opens new perspectives to design single-chain magnets.

Journal Article↗
PubMed1999

Evidence for dimerization of dimers in K+ channel assembly.

Voltage-gated K+ channels are tetrameric, but how the four subunits assemble is not known. We analyzed inactivation kinetics and peak current levels elicited for a variety of wild-type and mutant Kv1.3 subunits, expressed singly, in combination, and as tandem constructs, to show that 1) the dominant pathway involves a dimerization of dimers, and 2) dimer-dimer interaction may involve interaction sites that differ from those involved in monomer-monomer association. Moreover, using nondenaturing gel electrophoresis, we detected dimers and tetramers, but not trimers, in the translation reaction of Kv1.3 monomers.

Animals↗
PubMed2005

The role of the N-terminal heptad repeat of HIV-1 in the actual lipid mixing step as revealed by its substitution with distant coiled coils.

The gp41 glycoprotein of HIV-1 is considered to be responsible for the actual fusion process between the virus and the host membranes. According to a prevailing model, gp41 trimer organization, directed by the N-terminal coiled-coil region (NHR), is essential for steps involved in the actual merging of viral and cellular membranes. This study addresses a major question: Is the specific sequence of the NHR obligatory for the fusion process, or can it be replaced by distant coiled coils that form different oligomeric states in solution? For this purpose we synthesized three known GCN4 coiled-coil mutants that oligomerize in solution into either dimers, trimers, or tetramers. These peptides were chemically ligated to the fusion peptide thereby creating three chimera peptides with different oligomeric tendencies in solution. These peptides were investigated, together with the 70-mer wild-type peptide (N70), regarding their structure in solution and membrane by using circular dichroism (CD) and FTIR spectroscopies, their ability to induce vesicle fusion, and their ability to bind phospholipid membranes by using surface plasmon resonance (SPR). Our results suggest that local assembly of fusion peptides, facilitated by coiled-coil oligomers, increases lipid mixing ability, probably by facilitating stronger binding of the fusion peptides to the opposing membrane as revealed by SPR. However, N70 is significantly more active than the other chimeras. Overall, the data indicate a correlation between the distinct conformation of N70 in solution and in membranes and its enhanced lipid mixing relative to the GCN4 chimeras.

Amino Acid Sequence↗
PubMed1999

The C-terminal fragment of the precursor tail lysozyme of bacteriophage T4 stays as a structural component of the baseplate after cleavage.

Tail-associated lysozyme of bacteriophage T4 (tail lysozyme), the product of gene 5 (gp 5), is an essential structural component of the hub of the phage baseplate. It is synthesized as a 63-kDa precursor, which later cleaves to form mature gp 5 with a molecular weight of 43,000. To elucidate the role of the C-terminal region of the precursor protein, gene 5 was cloned and overexpressed and the product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, analytical ultracentrifugation, and circular dichroism. It was shown that the precursor protein tends to be cleaved into two fragments during expression and that the cleavage site is close to or perhaps identical to the cleavage site in the infected cell. The two fragments, however, remained associated. The lysozyme activity of the precursor or the nicked protein is about 10% of that of mature gp 5. Both the N-terminal mature tail lysozyme and the C-terminal fragment were then isolated and characterized by far-UV circular dichroism and analytical ultracentrifugation. The latter remained trimeric after dissociation from the N-terminal fragment and is rich in beta-structure as predicted by an empirical method. To trace the fate of the C-terminal fragment, antiserum was raised against a synthesized peptide of the last 12 C-terminal residues. Surprisingly, the C-terminal fragment was found in the tail and the phage particle by immunoblotting. The significance of this finding is discussed in relation to the molecular assembly and infection process.

Bacteriophage T4↗
PubMed1999

Characterization of TFG in mus musculus and Caenorhabditis elegans.

TFG was discovered as a fusion partner of NTRK1 in human papillary thyroid carcinoma. We assembled the mouse TFG cDNA from EST sequences and 5' end RACE product, identified full coding length TFG EST clones in pig (c17b07) and Schistosoma mansoni (SMNAS62), and analyzed the genomic structure of TFG in Caenorhabditis elegans (Y63D3A). The protein sequences of mouse, pig, and S. mansoni TFG are highly homologous to human TFG. The C. elegans sequence has diverged, but its predicted secondary structure is remarkably conserved. Human, mouse, and C. elegans TFG contain a putative trimeric N-terminal coiled-coil domain, glycosylation, myristylation, and phosphorylation sites, and SH2- and SH3-binding motifs. The SH2-binding motif is absent in C. elegans TFG. The expression of TFG does not vary among 7, 11, 15, and 19 day mouse embryonal stages. In situ hybridization with a TFG probe in 10, 5-day whole mouse embryos showed preferential staining of the limb buds, branchial arches, nasal processes, and brain, and weak staining of the primitive spinal cord and dorsal root ganglia.

Amino Acid Sequence↗
PubMed2004

Strand orientation by steric matching: a designed antiparallel coiled-coil trimer.

The design of an antiparallel coiled-coil 1:1:1 heterotrimer is described. Control of strand orientation results from proper alignment of sterically matched hydrophobic core side chains. Matched core layers position one cyclohexylalanine side chain against two alanine ones. Substitution of three consecutive heptad a positions with all permutations of two alanines and one cyclohexylalanine (AAX, AXA, AAX, where A = alanine, X = cyclohexylalanine) affords a parallel 1:1:1 heterotrimer, as previously reported. Here, we report that moving the substitution sites in one strand to d rather than a positions affords a new peptide that can form an antiparallel complex with the other original components. The new assembly is characterized by circular dichroism spectroscopy ([theta;](222) = -30 317 deg cm(2) dmol(-1), T(m) = 77 degrees C, DeltaG(unf) = 17.1 kcal/mol), and its stoichiometry and aggregation number are confirmed by nickel tag affinity analysis and analytical ultracentrifugation. Disulfide exchange data support the preference for an antiparallel arrangement. Examination of the functionally identical parallel complex demonstrates that the antiparallel structure is comparably stable, as confirmed by a direct competition assay that established an equilibrium 55:45 ratio of each assembly.

Alanine↗
PubMed1991

Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein.

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.

ADP-Ribosylation Factors↗
PubMed2005

Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization.

The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.

Bacteriophage P22↗
PubMed1990

Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.

Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.

Acetylglucosamine↗
PubMed2006

Liquid crystalline order from ortho-phenylene ethynylene macrocycles.

Triangular ortho-phenylene ethynylene (o-PE) cyclic trimers represent a novel member of shape-persistent macrocycles. Shape-persistent cyclic structures remain of great interest as molecular components in the fields of supramolecular materials, host-guest chemistry, and materials science. Novel discotic liquid crystalline properties are reported from triangular-shaped o-PE macrocycles containing branched alkoxy- and/or triethylene glycol (TEG) side chains using polarized optical microscopy (POM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The macrocycles self-assemble into thermotropic rectangular columnar (Colr) (for M1), hexagonal columnar (Colh) (for M2), and discotic nematic (for M3) mesophases at room temperature. This work shows clearly that electron-rich PE macrocycles can form LC materials. Alkyl side chains in M1 promote order, while hydrophilic side chains of M2 generate an amphiphilic structure that provides a different driving force for organization. The ability to create ordered self-assembling materials from these novel electron-rich macrocycles is important in nanotechnology.

Journal Article↗
PubMed2001

Heat shock factor-4 (HSF-4a) represses basal transcription through interaction with TFIIF.

The heat shock transcription factors (HSFs) regulate the expression of heat shock proteins (hsps), which are critical for normal cellular proliferation and differentiation. One of the HSFs, HSF-4, contains two alternative splice variants, one of which possesses transcriptional repressor properties in vivo. This repressor isoform inhibits basal transcription of hsps 27 and 90 in tissue culture cells. The molecular mechanisms of HSF-4a isoform-mediated transcriptional repression is unknown. Here, we present evidence that HSF-4a inhibits basal transcription in vivo when it is artificially targeted to basal promoters via the DNA-binding domain of the yeast transcription factor, GAL4. By using a highly purified, reconstituted in vitro transcription system, we show that HSF-4a represses basal transcription at an early step during preinitiation complex assembly, as pre-assembled preinitiation complexes are refractory to the inhibitory effect on transcription. This repression occurs by the HSF-4a isoform, but not by the HSF-4b isoform, which we show is capable of activating transcription from a heat shock element-driven promoter in vitro. The repression of basal transcription by HSF-4a occurs through interaction with the basal transcription factor TFIIF. TFIIF interacts with a segment of HSF-4a that is required for the trimerization of HSF-4a, and deletion of this segment no longer inhibits basal transcription. These studies suggest that HSF-4a inhibits basal transcription both in vivo and in vitro. Furthermore, this is the first report identifying an interaction between a transcriptional repressor with the basal transcription factor TFIIF.

Cell Line↗
PubMed2006

Carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization.

Coronavirus nucleocapsid (N) protein envelops the genomic RNA to form long helical nucleocapsid during virion assembly. Since N protein oligomerization is usually a crucial step in this process, characterization of such an oligomerization will help in the understanding of the possible mechanisms for nucleocapsid formation. The N protein of severe acute respiratory syndrome coronavirus (SARS-CoV) was recently discovered to self-associate by its carboxyl terminus. In this study, to further address the detailed understanding of the association feature of this C-terminus, its oligomerization was systematically investigated by size exclusion chromatography and chemical cross-linking assays. Our results clearly indicated that the C-terminal domain of SARS-CoV N protein could form not only dimers but also trimers, tetramers, and hexamers. Further analyses against six deletion mutants showed that residues 343-402 were necessary and sufficient for this C-terminus oligomerization. Although this segment contains many charged residues, differences in ionic strength have no effects on its oligomerization, indicating the absence of electrostatic force in SARS-CoV N protein C-terminus self-association. Gel shift assay results revealed that the SARS-CoV N protein C-terminus is also able to associate with nucleic acids and residues 363-382 are the responsible interaction partner, demonstrating that this fragment might involve genomic RNA binding sites. The fact that nucleic acid binding could promote the SARS-CoV N protein C-terminus to form high-order oligomers implies that the oligomeric SARS-CoV N protein probably combines with the viral genomic RNA in triggering long nucleocapsid formation.

Amino Acid Sequence↗
PubMed1994

Characterization of biosynthetic IgG oligomers resulting from light chain variable domain duplication.

We have characterized a human IgG1 monoclonal antibody composed of altered light chains. Each light chain consists of two identical variable domains and a kappa constant domain, in association with a normal gamma chain. This antibody assembled biosynthetically into a mixture of stable oligomers and monomers. Employing gel filtration, PAGE, and electron microscopy, we examined the antibody and the nature of the associations involved in oligomer formation. By engineering a protease factor Xa site between the duplicated light chain variable domains and examining the fragments produced following factor Xa cleavage, we demonstrated the association of the IgG monomers occurred through their duplicated VL domains. Electron microscopy showed the oligomeric antibody to be predominantly dimers and trimers in which the monomeric units were associated through the tips of the Fab portion of the antibody, presumably through the protruding N-terminal VL domains. Similar examination of monomers demonstrated several molecular forms, including individual molecules with self-crosslinked Fab arms and others displaying the open Y and T shapes typically observed for IgG antibodies. The monomers also displayed distally protruding domain-like structures. The oligomers produced by this cell line therefore occurred through the noncovalent interaction between the extra light chain variable domains.

Amino Acid Sequence↗
PubMed2003

Specific control of peptide assembly with combined hydrophilic and hydrophobic interfaces.

Designed coiled-coil heterotrimers are described whose assembly is governed by both hydrophobic and hydrophilic forces. Sterically matched hydrophobic core side-chain packing of alanine and cyclohexylalanine has been shown to promote formation of a 1:1:1 heterotrimer. Manipulation of hydrophilic glutamic acid (Glu)/lysine (Lys) pairs at each of three helical contact interfaces provides a secondary recognition mechanism. Peptides with matched cores and hydrophilic contacts form stable heterotrimers (DeltaG(unf) at 25 degrees C = 17.93 kcal/mol; MW(app) = 11362 vs 11563 calcd for trimer), as do those with a single Lys/Lys (but not Glu/Glu) interface. The additional specificity engendered by simultaneous operation of two interfaces was used to design a system in which six different peptides are mixed to form three specific and independent heterotrimers in the same solution.

Alanine↗