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At least 217 records · Page 12Linked to original sources
PubMed1983

Analysis of Ad5 hexon and 100K ts mutants using conformation-specific monoclonal antibodies.

Adenovirus type 5 ts mutants deficient in hexon metabolism were investigated using conformation-specific monoclonal antibodies directed against hexon capsomeres and the viral 100K protein. The ts mutants map either in the hexon structural gene or in the gene encoding the 100K protein, a major, late nonstructural protein. All of the mutants examined (ts1, ts2, ts3, ts4, ts17, and ts20 of J. F. Williams, M. Gharpure, S. Ustacelebi, and S. McDonald (1971). J. Gen. Virol. 11, 95-101) were unable to produce the capsomeric form of hexon (a trimer of three hexon monomers) at the nonpermissive temperature. However, all of the mutants retained the ability to produce a complex of 100K and hexon which has been demonstrated to play a major role in the assembly of hexon trimers. The mutants accumulated nontrimerized hexon in this ts complex in the perinuclear region of the cell. Several of the mutants (ts1, ts2, ts3) were found to successfully assemble hexon synthesized at the nonpermissive temperature upon shift down to the permissive temperature, even in the presence of a protein synthesis inhibitor. The mutant, ts2, which maps in the hexon structural gene, was found to be dependent on protein synthesis for transport of hexon trimers into the nucleus during temperature shift down, while the 100K ts mutants, ts1 and ts3, were independent of protein synthesis for both hexon assembly and transport.

Adenoviruses, Human↗
PubMed1991

Synthesis of truncated amino-terminal trimers of thrombospondin.

Thrombospondin (TSP) is a 450-kDa glycoprotein that is comprised of three identical disulfide-bonded subunits (1152 amino acids) held together near the heparin-binding amino-terminal globular domains. TSP truncated at residue 277 (TSP-277) or 381 (TSP-381) consisted largely of disulfide-bonded trimers when expressed in COS cells or insect cells. In addition, TSP-381 formed heterotrimers with endogenous COS cell TSP. Cleavage of TSP and the truncated mutants in the proteolytically sensitive region between residues 220 and 237 yielded monomeric amino-terminal fragments. Cys-252 and Cys-256 are the only cysteines between residues 238 and 277 and therefore must bridge among subunits. TSP-381 in which Cys-252 and Cys-256 were changed to glycine was secreted efficiently by COS cells but with only a minor portion of the protein in the form of disulfide-bonded trimers. The sequence of TSP between residues 258 and 283 is predicted to form an amphiphatic alpha-helix. We suggest that assembly of TSP trimers involves formation of an alpha-helical coiled-coil structure which is stabilized by formation of disulfides.

Amino Acid Sequence↗
PubMed2004

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle.

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3' and 5' ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.

Base Sequence↗
PubMed1996

Site-directed mutagenesis of Cys-15 and Cys-20 of pulmonary surfactant protein D. Expression of a trimeric protein with altered anti-viral properties.

Surfactant protein D (SP-D) molecules are preferentially assembled as dodecamers consisting of trimeric subunits associated at their amino termini. The NH2-terminal sequence of each monomer contains two conserved cysteine residues, which participate in interchain disulfide bonds. In order to study the roles of these residues in SP-D assembly and function, we employed site-directed mutagenesis to substitute serine for cysteine 15 and 20 in recombinant rat SP-D (RrSP-D), and have expressed the mutant (RrSP-Dser15/20) in Chinese hamster ovary (CHO-K1) cells. The mutant, which was efficiently secreted, bound to maltosyl-agarose, but unlike RrSP-D, was assembled exclusively as trimers. The constituent monomers showed a decreased mobility on SDS-polyacrylamide gel electrophoresis resulting from an increase in the size and sialylation of the N-linked oligosaccharide at Asn-70. Although RrSP-Dser15/20 contained a pepsin-resistant triple helical domain, it showed a decreased Tm, and acquired susceptibility to proteolytic degradation. Like RrSP-D, RrSP-Dser15/20 bound to the hemagglutinin of influenza A. However, it showed no viral aggregation and did not enhance the binding of influenza A to neutrophils (PMN), augment PMN respiratory burst, or protect PMNs from deactivation. These studies indicate that amino-terminal disulfides are required to stabilize dodecamers, and support our hypothesis that the oligomerization of trimeric subunits contributes to the anti-microbial properties of SP-D.

Animals↗
PubMed2006

Trimeric structure of the wild soluble chloride intracellular ion channel CLIC4 observed in crystals.

The crystal structure of a wild type of the human soluble chloride intracellular ion channel CLIC4 (wCLIC4) has been determined at a resolution of 2.2A. The structure shows a homotrimer in an asymmetric unit, which is first observed in CLICs. The assembly of the trimer takes a unique triple interaction mode between three monomers with a hydrogen-bond network and hydrophobic contacts. Through such complicated interactions, the homotrimer of wCLIC4 is firmly stabilized. The structure shows an oligomeric mode with a unique assembly mechanism by which the oligomerization of CLIC4 can be performed without any intramolecular disulfide bond formation. It indicated a possibility that CLIC4 may take a unique structural organization distinct from CLIC1 for docking with lipid bilayers. In addition, the structure shows distinct conformational states of the h2 region for respective monomers of the trimer, which reveal an intrinsic conformational susceptibility for this significant region in the structural transition.

Amino Acid Sequence↗
PubMed2006

Controlled assembly of a heterogeneous single-site ethylene trimerization catalyst as probed by X-ray absorption spectroscopy.

X-ray absorption spectroscopy at the Cr K- and L(2,3)-edges was used to study the assembling process of a heterogeneous Cr-based single-site catalyst. The starting point was a Phillips-type system with monochromate species anchored on a silica surface, which was first reduced to a variety of different surface Cr(II) species. The reduced sample was modified with a 1,3,5-tribenzylhexahydro 1,3,5-triazine (TAC) ligand in the presence of CH(2)Cl(2) as solvent to yield a heterogeneous single-site Cr-based catalyst active in the trimerization of ethylene. The molecular structure of the resultant catalytic material consists of distorted octahedral Cr(III) species. The extended X-ray absorption fine-structure (EXAFS) spectroscopy fitting procedure in R space up to 2.5 A showed that the synthesis leads to coordination with a TAC ligand. The fit also shows that it was possible to complete the six-fold environment around Cr(III) with two oxygen atoms and one chloride ligand. This chloride ligand is formed in a redox process from the solvent and is responsible for the oxidation of surface Cr(II) to Cr(III). The obtained geometry and the local environment of the surface complex are discussed in light of its homogeneous counterpart and confirm the single-site characteristics of the prepared catalytic material.

Journal Article↗
PubMed2002

Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein.

P22 tailspike is a homotrimeric, thermostable adhesin that recognizes the O-antigen lipopolysaccharide of Salmonella typhimurium. The 70 kDa subunits include long beta-helix domains. After residue 540, the polypeptide chains change their path and wrap around one another, with extensive interchain contacts. Formation of this interdigitated domain intimately couples the chain folding and assembly mechanisms. The earliest detectable trimeric intermediate in the tailspike folding and assembly pathway is the protrimer, suspected to be a precursor of the native trimer structure. We have directly analyzed the kinetics of in vitro protrimer formation and disappearance for wild type and mutant tailspike proteins. The results confirm that the protrimer intermediate is an on-pathway intermediate for tailspike folding. Protrimer was originally resolved during tailspike folding because its migration through nondenaturing polyacrylamide gels was significantly retarded with respect to the migration of the native tailspike trimer. By comparing protein mobility versus acrylamide concentration, we find that the retarded mobility of the protrimer is due exclusively to a larger overall size than the native trimer, rather than an altered net surface charge. Experiments with mutant tailspike proteins indicate that the conformation difference between protrimer and native tailspike trimer is localized toward the C-termini of the tailspike polypeptide chains. These results suggest that the transformation of the protrimer to the native tailspike trimer represents the C-terminal interdigitation of the three polypeptide chains. This late step may confer the detergent-resistance, protease-resistance, and thermostability of the native trimer.

Amino Acid Substitution↗
PubMed1996

Assembly-defective OmpC mutants of Escherichia coli K-12.

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.

Alleles↗
PubMed2004

Zinc binding drives the folding and association of the homo-trimeric gamma-carbonic anhydrase from Methanosarcina thermophila.

Carbonic anhydrase from the archeon Methanosarcina thermophila (Cam) is a homo-trimeric enzyme, the left-handed beta-helical subunits of which bind three catalytic Zn(2+) ions at symmetry-related subunit interfaces. The observation of activity for holo-Cam at nanomolar concentrations provides a minimal estimated free energy of folding and assembly of the trimeric holo-complex of approximately 70 kcal (mol trimer)(-1) at standard state. Although the direct measurement of stability by chemical denaturation was precluded by the irreversible unfolding of the holo-enzyme, the reversible unfolding of metal-free apo-Cam is well described by a three-state model involving the folded apo-trimer, the folded monomer and the unfolded monomer. The monomer is estimated to have a stability of 4.0 +/- 0.3 kcal (mol monomer)(-1). The association to form apo-trimer contributes 13.2 +/- 0.4 kcal (mol trimer)(-1), a value confirmed by analytical ultracentrifugation measurements. Far- and near-UV circular dichroism data show a progressive increase in secondary and tertiary structure as the apo-monomer is converted to holo-trimer. The literature value for the free energy of binding of one Zn(2+) ion to a canonical active site, 16.4 kcal mol(-1), is consistent with the presumption that the >45 kcal (mol trimer)(-1) generated by the binding of three ions represents the major contribution to the stability of the holo-trimeric Cam.

Binding Sites↗
PubMed2002

Insight into Schmid metaphyseal chondrodysplasia from the crystal structure of the collagen X NC1 domain trimer.

Collagen X is expressed specifically in the growth plate of long bones. Its C1q-like C-terminal NC1 domain forms a stable homotrimer and is crucial for collagen X assembly. Mutations in the NC1 domain cause Schmid metaphyseal chondrodysplasia (SMCD). The crystal structure at 2.0 A resolution of the human collagen X NC1 domain reveals an intimate trimeric assembly strengthened by a buried cluster of calcium ions. Three strips of exposed aromatic residues on the surface of NC1 trimer are likely to be involved in the supramolecular assembly of collagen X. Most internal SMCD mutations probably prevent protein folding, whereas mutations of surface residues may affect the collagen X suprastructure in a dominant-negative manner.

Amino Acid Sequence↗
PubMedDate not supplied

A genetic engineering approach to study the mode of assembly of the OmpF porin in the envelope of E coli.

Inducible hybrid genes encoding two large domains, a periplasmic domain consisting of the PhoS sequence and an outer membrane domain corresponding to various lengths of the OmpF mature sequence were constructed. The synthesized hybrid polypeptides are correctly processed during the early times of induction, their precursor forms being accumulated at later times. These hybrids restore sensitivity toward colicin A to ompF E coli B strain which suggests an outer membrane location. At least 2 of them are indeed localized in the outer membrane after immunogold labelling on ultrathin cryosections. Insertion of a hydrophobic sequence between PhoS and OmpF improves the trimerization and the assembly of the OmpF part. Only the hybrids presenting the last C-terminal 29 residues of OmpF are able to promote the colicin N killing action and to exhibit a trimeric conformation which is recognized by specific antibodies. Moreover, the deletion of the C-terminal region impairs the functional insertion of the OmpF domain; this indicates that the last membrane-spanning region of OmpF is necessary for the correct folding and orientation of the protein in the outer membrane.

Antibodies, Monoclonal↗
PubMed1983

Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme.

Extracts of liver from hemizygous affected mice with the X-linked spfash mutation have 5 to 10 percent of normal ornithine transcarbamylase (OTC) activity, yet the homogeneous enzyme isolated from these extracts is identical to that in controls. The OTC messenger RNA from mutant livers programs the synthesis of two distinct OTC precursor polypeptides--one normal in size, the other distinctly elongated. Both precursors are imported and proteolytically processed by mitochondria, but only the normal one is assembled into active trimer. This novel phenotype may result from a mutation in the structural gene for OTC leading, primarily, to aberrant splicing of OTC messenger RNA and, secondarily, to formation of a structurally altered precursor whose posttranslational pathway is ultimately futile because its mature mitochondrial form is not capable of assembly and functional expression.

Animals↗
PubMed2003

Identification of a heparin-binding motif on adeno-associated virus type 2 capsids.

Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary.

Amino Acid Motifs↗
PubMed1990

Adenovirus serotype 3 fibre protein is expressed as a trimer in Escherichia coli.

The adenovirus serotype 3 (Ad3) fibre has been expressed in Escherichia coli as an insoluble protein. The protein was solubilized by extraction with urea. Slow removal of urea during the purification procedure resulted in a soluble Ad3 fibre preparation. Polyacrylamide gel analysis of the purified fibre protein, as well as cross-linking experiments performed on cellular debris of expressing cells, suggest that the recombinant Ad3 fibre self-assembles as a trimer from identical polypeptide chains. Gel filtration gave the same exclusion volume for the purified recombinant fibre and for the native fibre in the protein mixture extracted from the Ad3-infected cells. The recombinant fibre was partially resistant to proteolytic degradation, suggesting a folded structure.

Adenoviridae↗
PubMed2004

A model of a transmembrane drug-efflux pump from Gram-negative bacteria.

In Gram-negative bacteria, drug resistance is due in part to the activity of transmembrane efflux-pumps, which are composed of three types of proteins. A representative pump from Escherichia coli is an assembly of the trimeric outer-membrane protein TolC, which is an allosteric channel, the trimeric inner-membrane proton-antiporter AcrB, and the periplasmic protein, AcrA. The pump displaces drugs vectorially from the bacterium using proton electrochemical force. Crystal structures are available for TolC and AcrB from E. coli, and for the AcrA homologue MexA from Pseudomonas aeruginosa. Based on homology modelling and molecular docking, we show how AcrA, AcrB and TolC might assemble to form a tripartite pump, and how allostery may occur during transport.

Bacterial Outer Membrane Proteins↗
PubMed1982

Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.

Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed2000

Overexpression of protease-deficient DegP(S210A) rescues the lethal phenotype of Escherichia coli OmpF assembly mutants in a degP background.

Replacement of OmpF's conserved carboxy-terminal phenylalanine with dissimilar amino acids severely impaired its assembly into stable trimers. In some instances, interactions of mutant proteins with the outer membrane were also affected, as judged by their hypersensitivity phenotype. Synthesis of all mutant OmpF proteins elevated the expression of periplasmic protease DegP, and synthesis of most of them made its presence obligatory for cell viability. These results showed a critical role for DegP in the event of aberrant outer membrane protein assembly. The lethal phenotype of mutant OmpF proteins in a degP null background was eliminated when a protease-deficient DegP(S210A) protein was overproduced. Our data showed that this rescue from lethality and a subsequent increase in mutant protein levels in the envelope did not lead to the proper assembly of the mutant proteins in the outer membrane. Rather, a detergent-soluble and thermolabile OmpF species resembling monomers accumulated in the mutants, and to a lesser extent in the parental strain, when DegP(S210A) was overproduced. Interestingly, this also led to the localization of a significant amount of mutant polypeptides to the inner membrane, where DegP(S210A) also fractionated. These results suggested that the DegP(S210A)-mediated rescue from toxicity involved preferential sequestration of misfolded OmpF monomers from the normal assembly pathway.

Amino Acid Substitution↗
PubMed2003

The roles of toc34 and toc75 in targeting the toc159 preprotein receptor to chloroplasts.

The Toc complex at the outer envelope of chloroplasts initiates the import of nuclear-encoded preproteins from the cytosol into the organelle. The core of the Toc complex is composed of two receptor GTPases, Toc159 and Toc34, as well as Toc75, a beta-barrel membrane channel. Toc159 is equally distributed between a soluble cytoplasmic form and a membrane-inserted form, suggesting that assembly of the Toc complex is dynamic. In the present study, we used the Arabidopsis thaliana orthologs of Toc159 and Toc34, atToc159 and atToc33, respectively, to investigate the requirements for assembly of the trimeric Toc complex. In addition to its intrinsic GTPase activity, we demonstrate that integration of atToc159 into the Toc complex requires atToc33 GTPase activity. Additionally, we show that the interaction of the two GTPase domains stimulates association of the membrane anchor of atToc159 with the translocon. Finally, we employ reconstituted proteoliposomes to demonstrate that proper insertion of the receptor requires both Toc75 and Toc34. Collectively these data suggest that Toc34 and Toc75 act sequentially to mediate docking and insertion of Toc159 resulting in assembly of the functional translocon.

Arabidopsis↗