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PubMed2003

[Mn2(saltmen)2Ni(pao)2(L)2](A)2 with L=pyridine, 4-picoline, 4-tert-butylpyridine, N-methylimidazole and A=ClO4-, BF4-, PF6-, ReO4-: a family of single-chain magnets.

A series of single-chain magnets, [Mn2(saltmen)2Ni(pao)2(L)2](A)2 (saltmen(2-)=N,N'-(1,1,2,2-tetramethylethylene) bis(salicylideneiminate), pao-=pyridine-2-aldoximate; A-=ClO4- with L=4-picoline; 2, 4-tert-butylpyridine; 3, N-methylimidazole; 4, and L=pyridine with A-=BF4-; 5, PF6-; 6, ReO4-; 7), was prepared by reactions between MnIII dimer units, i.e., [Mn2(saltmen)2(H2O)2](A)2 (A-=ClO4-, BF4-, PF6-) or Mn2(saltmen)2(ReO4)2, and NiII monomeric units, i.e., Ni(pao)2(L)2, in methanol/water media. The crystal structures of 4, 6, and 7 were established by single-crystal X-ray crystallography. These three compounds are isostructural with [Mn2(saltmen)2Ni(pao)2(py)2](ClO4)2 (1) (Clérac, R.; Miyasaka, H.; Yamashita, M.; Coulon, C. J. Am. Chem. Soc. 2002, 124, 12837) and crystallize in monoclinic space group C2/c. The linear arrangement of MnIII dimer units and NiII building blocks leads to an alternating chain having a repeating unit, [-(O)2-Mn-ON-Ni-NO-Mn-]. The chains are well separated with the nearest interchain intermetallic distance of 10.36 A for 4, 10.51 A for 6, and 10.30 A for 7, and there is no significant pi-pi interchain interaction between ligands. The void space between the chains is occupied by counteranions, which control the three-dimensional organization of the chains. The X-ray diffraction analysis (XRD) on a powder sample was also performed for all compounds. The XRD patterns for 1, 2, and 4-7 are very similar, emphasizing the isostructural nature of these materials although they have individually slight different interchain distances. Inversely, the XRD pattern for 3 reveals a completely different shape being indicative of the peculiar crystal packing compared to the others. Nevertheless, the one-dimensional nature of the structure is also kept in 3 as indicated by magnetic measurements. The whole family of compounds exhibits quasi-identical magnetic behavior compared to that described for 1. Above 30 K, the heterometallic chain can be described as an assembly of antiferromagnetically coupled Mn...Ni....Mn trimers (via oximate bridge, -24.2 K<J(Mn-Ni)/kB<-20.8 K) connected through Mn...Mn ferromagnetic interaction (via the phenolate bridge, J(Mn-Mn)/kB approximately +0.7 K), assuming a ferromagnetic chain with S=3 units. In the low temperature region, combined ac and dc magnetic measurements revealed for the whole series the systematic presence of a magnet behavior exhibiting coercivity and slow relaxation of magnetization below 3.5 K. This behavior was analyzed on the basis of Glauber's theory for an Ising one-dimensional system which predicts an activated dependence (Arrhenius law) of the relaxation time: tau=tau0 exp(Delta/kBT). Similar values of tau0 approximately 1x10(-10) s and Delta/kB approximately 70 K have been found along the series. The described compounds constitute the first example of the rational design of a single-chain magnet family and clearly demonstrate the unique behavior of these heterometallic chains independently of their interchain environments.

Journal Article↗
PubMed2003

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of metal-ion selected dynamic protein libraries.

The application of electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry to the investigation of the relative stabilities (and thus packing efficiencies) of Fe-bound trihelix peptide bundles is demonstrated. Small dynamic protein libraries are created by metal-ion assisted assembly of peptide subunits. Control of the trimeric aggregation state is coupled to stability selection by exploiting the coordination requirements of Fe(2+) in the presence of bidentate 2,2'-bipyridyl ligands covalently appended to the peptide monomers. At limiting metal-ion concentration, the most thermodynamically stable, optimally packed peptide trimers dominate the mass spectrum. The identities of optimally stable candidate trimers observed in the ESI FT-ICR mass spectra are confirmed by resynthesis of exchange-inert analogues and measurement of their folding free energies. The peptide composition of the trimers may be determined by infrared multiphoton dissociation (IRMPD) MS(3) experiments. Additional sequence information for the peptide subunits is obtained from electron capture dissociation (ECD) of peptides and metal-bound trimers. The experiments also suggest the presence of secondary structure in the gas phase, possibly due to partial retention of the solution-phase coiled coil structure.

Amino Acid Sequence↗
PubMed2005

Structure, assembly, and topology of the G185R mutant of the fourth transmembrane domain of divalent metal transporter.

The mammalian iron transporter, divalent metal transporter (DMT1), is a 12-transmembrane domain integral protein, responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Two disease-causing mutants in animals have been found and attributed to the same missense mutation (G185R), which occurs within the putative transmembrane domain 4 (TM4) of DMT1. We have characterized a synthetic 24-mer peptide, corresponding to the sequence of the TM4 of DMT1 with G185R mutation using circular dichroism (CD) and NMR spectroscopy and show that the G185R peptide assumes mainly alpha-helical conformations in various membrane-mimetic environments. Solution structures derived from NMR and molecular dynamics/simulated annealing calculations demonstrate that the peptide exhibits a highly defined alpha-helix in its middle portion, flanked by a highly flexible N-terminus and a relatively ordered C-terminus. Both the folding and location of the C-terminus in SDS micelles are regulated by pH values. Paramagnetic broadening on peptide NMR signals by spin-labeled 5- and 16-doxylstearic acids and Mn(2+) ion suggests that both the N-terminus and the helical region of the peptide are embedded in SDS micelles. Surprisingly, self-association of the peptides for both the wild type and the G185R mutant studied by CD, electrospray ionization mass spectrometry, and NMR diffusion-ordered spectroscopy demonstrated that mutation of the Gly185 to a bulky and positively charged arginine causes a different self-assembly of the peptide, e.g., from a trimer to a hexamer, which implies that the quaternary structure of integral DMT1 may be crucial for its function in vivo.

Amino Acid Sequence↗
PubMed1997

Disulphide-bonded intermediate on the folding and assembly pathway of a non-disulphide bonded protein.

The trimeric parallel beta-coil P22 tailspike contains eight cysteines per chain, but lacks disulphide bonds in the native state, in both the crystalline and solution forms. However, cysteines in a folding intermediate are reactive with thiol blocking reagents, which prevent further productive folding both in vivo and in vitro. The in vivo refolding yield was independent of the availability of metal ions, but was sensitive to redox potential. Isolation by nondenaturing gel electrophoresis of the protrimer intermediate, a trimeric folding intermediate that precedes the fully folded trimer in the in vivo and in vitro pathways, revealed the presence of interchain disulphide bonds. Incubation of the isolated protrimer with reducing agents generated the native trimer. The formation of beta-sheets with interdigitated strands from different subunits in the native trimer may require the transient disulphide bonds for proper alignment. To our knowledge this is the first report of a disulphide bond present in a folding intermediate of a non-disulphide bonded protein.

Cysteine↗
PubMed1993

Reconstitution of the folding pathway of collagen in a cell-free system: formation of correctly aligned and hydroxylated triple helices.

We describe here a cell-free system which will carry out the initial stages in the synthesis, post-translational modification and assembly of type-X collagen. The mRNA coding for bovine type-X collagen was synthesized in vitro and translated in a rabbit reticulocyte lysate to yield a protein that was collagenase sensitive and could be immunoprecipitated with antibodies raised to purified avian type-X collagen. When type-X collagen was synthesized in the absence of added microsomes or in the presence of canine pancreas microsomes, the translation products showed partial resistance to digestion with pepsin but were completely degraded with a mixture of chymotrypsin and trypsin, suggesting that only incorrectly aligned non-native collagen molecules were synthesized under these conditions. When the protein was synthesized in the presence of microsomes derived from avian fibroblasts or a human fibrosarcoma cell line, the translocated product migrated as a diffuse band characteristic of hydroxylated collagen. The synthesized polypeptides were also resistant to both pepsin and trypsin/chymotrypsin digestion, demonstrating the formation of correctly aligned native collagen. Furthermore, the collagen polypeptides assembled into higher-order structures, possibly trimers, which were stabilized by interchain disulphide bonds. The collagen helix synthesized in vitro had a melting temperature of 41 degrees C which is comparable with the protein synthesized in vivo, further demonstrating that the polypeptides were hydroxylated and that the triple helix formed was correctly aligned.

Animals↗
PubMed2003

Selective heteromeric assembly of cyclic nucleotide-gated channels.

Many ion channels in vivo are heteromeric complexes with well defined subunit compositions. For some channels, domains have been identified that determine whether two or more subunit species are compatible in forming a complex. Nonetheless, an unsolved fundamental question is how the native composition of an ion channel is selected during assembly over functional alternatives, such as heteromeric complexes favored over homomers. Cyclic nucleotide-gated channels are tetramers and, in their native forms, are composed of A and B subunits. Although most A subunits can form functional homomeric channels when expressed alone, A/B heteromeric channels are selectively formed in the presence of a B subunit. Here, we show that this selective assembly of heteromeric channels requires a trimer-forming C-terminal leucine zipper (CLZ) domain recently identified in the distal C terminus of A, but not B, subunits. Thus, a CLZ-defective A subunit no longer forms predominantly A/B heteromeric channels with the B subunit. A mechanism for this specificity involving the trimerization of the CLZ domain is proposed.

Amino Acid Sequence↗
PubMed2004

The crystal structure of Escherichia coli MoaB suggests a probable role in molybdenum cofactor synthesis.

The crystal structure of Escherichia coli MoaB was determined by multiwavelength anomalous diffraction phasing and refined at 1.6-A resolution. The molecule displayed a modified Rossman fold. MoaB is assembled into a hexamer composed of two trimers. The monomers have high structural similarity with two proteins, MogA and MoeA, from the molybdenum cofactor synthesis pathway in E. coli, as well as with domains of mammalian gephyrin and plant Cnx1, which are also involved in molybdopterin synthesis. Structural comparison between these proteins and the amino acid conservation patterns revealed a putative active site in MoaB. The structural analysis of this site allowed to advance several hypothesis that can be tested in further studies.

Arabidopsis Proteins↗
PubMed2006

Crystal structures of saposins A and C.

Saposins A and C are sphingolipid activator proteins required for the lysosomal breakdown of galactosylceramide and glucosylceramide, respectively. The saposins interact with lipids, leading to an enhanced accessibility of the lipid headgroups to their cognate hydrolases. We have determined the crystal structures of human saposins A and C to 2.0 Angstroms and 2.4 Angstroms, respectively, and both reveal the compact, monomeric saposin fold. We confirmed that these two proteins were monomeric in solution at pH 7.0 by analytical centrifugation. However, at pH 4.8, in the presence of the detergent C(8)E(5), saposin A assembled into dimers, while saposin C formed trimers. Saposin B was dimeric under all conditions tested. The self-association of the saposins is likely to be relevant to how these small proteins interact with lipids, membranes, and hydrolase enzymes.

Amino Acid Sequence↗
PubMed2004

Disulfide-dependent multimeric assembly of resistin family hormones.

Resistin, founding member of the resistin-like molecule (RELM) hormone family, is secreted selectively from adipocytes and induces liver-specific antagonism of insulin action, thus providing a potential molecular link between obesity and diabetes. Crystal structures of resistin and RELMbeta reveal an unusual multimeric structure. Each protomer comprises a carboxy-terminal disulfide-rich beta-sandwich "head" domain and an amino-terminal alpha-helical "tail" segment. The alpha-helical segments associate to form three-stranded coiled coils, and surface-exposed interchain disulfide linkages mediate the formation of tail-to-tail hexamers. Analysis of serum samples shows that resistin circulates in two distinct assembly states, likely corresponding to hexamers and trimers. Infusion of a resistin mutant, lacking the intertrimer disulfide bonds, in pancreatic-insulin clamp studies reveals substantially more potent effects on hepatic insulin sensitivity than those observed with wild-type resistin. This result suggests that processing of the intertrimer disulfide bonds may reflect an obligatory step toward activation.

Adipocytes↗
PubMed1988

Folding, trimerization, and transport are sequential events in the biogenesis of influenza virus hemagglutinin.

Results from several systems indicate that correct protein folding and subunit assembly correlate with the transport of membrane and secretory proteins from the endoplasmic reticulum (ER) to the Golgi complex. Because the site of oligomer assembly and its precise relationship to intracellular transport remain unclear, we have studied in detail the folding and trimerization of the influenza virus hemagglutinin (HA0) relative to its transport from ER to Golgi. Trimerization and transport were analyzed using several different methods, including transport inhibitors, temperature blocks, semi-intact cells, in vitro protein translocation, and immunocytochemistry. Taken together, the results clearly demonstrate that trimerization occurs at a point prior to exit from the ER. Before assembly, HA0 monomers were extensively folded and possessed intramolecular disulfide bonds, but monomers were not transported to the cis Golgi compartment. Thus, hemagglutinin progresses through at least two intermediate states before transport to the Golgi: highly folded monomers and trimers that have not yet left the ER.

Adenosine Triphosphate↗
PubMed2004

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid.

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles.

Amino Acid Substitution↗
PubMed1995

Requirement of ATP hydrolysis for assembly of ClpA/ClpP complex, the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

Adenosine Triphosphatases↗
PubMed1988

Self-assembly of apoferritin from horse spleen after reversible chemical modification with 2,3-dimethylmaleic anhydride.

Apoferritin from horse spleen is composed of 24 subunits that undergo partial dissociation after chemical modification with 2,3-dimethylmaleic anhydride (DMMA), yielding dimeric, trimeric, and tetrameric intermediates, stable at pH 8.5 and 0 degrees C. Deacylation at neutral pH and elevated temperature provides a means to initiate reassembly by appropriate shifts of the solvent conditions. In order to monitor the pathway of self-assembly, starting from different intermediates of dissociation, dimers, trimers, and tetramers were isolated and investigated with respect to their capacity to accomplish reassociation. Intrinsic protein fluorescence, gel permeation chromatography, and analytical ultracentrifugation were applied to characterize the intermediate and final stages of association. The assembly of both the dimer and trimer yields greater than 85% of the native tetracosamer; the overall rate, starting from the dimer, exceeds the one starting from the trimer. Under comparable conditions, the tetramer exhibits only partial reassociation via the dimer and monomer; the corresponding dissociation reaction determines the observed slower rate. Significant assembly intermediates are "structured monomers", dimers, trimers, and dodecamers. Polymerization of the dimer via the tetramer, octamer, etc., does not occur on the pathway of assembly. The results confirm the assembly scheme proposed previously on the basis of cross-linking and spectroscopic experiments [Gerl, M., & Jaenicke, R. (1987) Eur. Biophys. J. 15, 103-109]. Comparison of structural models involving the different subunit interactions responsible for the sequential association supports the monomer----dimer----trimer----hexamer----dodecamer----tetracosamer mechanism of apoferritin self-assembly.

Animals↗
PubMed1993

Dynamic equilibrium between vesicular stomatitis virus glycoprotein monomers and trimers in the Golgi and at the cell surface.

Previous studies have shown that trimers of the vesicular stomatitis virus glycoprotein (VSV G protein) are in rapid equilibrium with monomeric subunits after folding and assembly in the endoplasmic reticulum (ER). To determine whether G protein trimers were in equilibrium with monomers in other cellular compartments, we studied heterotrimer formation between VSV G protein and a mutant G protein (G mu protein) containing a 3-amino-acid cytoplasmic domain replacing the normal 29-amino-acid domain. The G mu protein is transported from the ER much more slowly than G protein, although both G and G mu proteins form trimers rapidly in the ER. In coexpression experiments, we observed that VSV G protein molecules exited the ER about sixfold faster than G mu protein molecules, and we observed no heterotrimer formation in the ER, probably because of rapid reassortment of the mutant and wild-type trimers. However, heterotrimer formation between the two proteins was observed after long chase periods that allowed time for trimers of the mutant protein to reach the plasma membrane and reassort with the G protein subunits. Additional studies showed that heterotrimers of the two proteins could form in the Golgi or in the ER if exit of the G protein from either compartment was blocked.

Animals↗
PubMed1998

Engineering trimeric fibrous proteins based on bacteriophage T4 adhesins.

The adsorption specificity of bacteriophage T4 is determined by genes 12 and 37, encoding the short tail-fibers (STF) and the distal part of the long tail-fibers (LTF), respectively. Both are trimeric proteins with rod domains made up of similar tandem quasi-repeats, approximately 40 amino acids long. Their assembly requires the viral chaperones gp57A and gp38. Here we report that fusing fragments of gp12 and gp37 to another trimeric T4 fibrous protein, fibritin, facilitates correct assembly, thereby by-passing the chaperone requirement. Fibritin is an alpha-helical coiled coil protein whose C-terminal part (fibritin E, comprising the last 120 residues) has recently been solved to atomic resolution. Gp12 fragments of 109 and 70 amino acids, corresponding to three and two quasi-repeats respectively, were fused to the C-terminus of fibritin E. A similar chimera was designed for the last 63 residues of gp37, which contain four copies of the pentapeptide Gly-X-His-X-His and assume a narrow rigid structure in the LTF distal tip. Expressed from plasmids, all three chimeras form soluble trimers that are resistant to dissociation by SDS and digestion by trypsin, indicative of correct folding and oligomerization.

Amino Acid Sequence↗
PubMed2004

Gamma-secretase complex assembly within the early secretory pathway.

gamma-Secretase is an aspartyl protease complex composed of the four core components APH-1, nicastrin (NCT), presenilin (PS), and PEN-2. It catalyzes the final intramembranous cleavage of the beta-secretase-processed beta-amyloid precursor protein to liberate the neurotoxic amyloid beta-peptide. Whereas unassembled complex components appear to be unstable and/or to be retained within the endoplasmic reticulum (ER), the fully assembled complex is known to exert its biological function in late secretory compartments, including the plasma membrane. We thus hypothesized that the gamma-secretase complex undergoes a stepwise assembly within the ER. We demonstrate that gamma-secretase-associated NCT can be actively retained within the ER by the addition of a retention signal. Under these conditions, complex assembly occurred in the absence of maturation of NCT, and ER-retained immature NCT associated with APH-1, PEN-2, and PS fragments. Moreover, a biotinylated transition state gamma-secretase inhibitor allowed the preferential isolation of the fully assembled complex containing immature NCT. Furthermore, we observed a conformational change in immature NCT, which is known to be selectively associated with complete gamma-secretase complex assembly. This was also observed for a small amount of immature endogenous NCT. ER-retained NCT also rescued the biochemical phenotype observed upon RNA interference-mediated NCT knockdown, viz. reduced amyloid beta-peptide production; instability of PS, PEN-2, and APH-1; and accumulation of beta-amyloid precursor protein C-terminal fragments. Finally, we demonstrate that dimeric (NCT/APH-1) and trimeric (NCT/APH-1/PS) intermediates of gamma-secretase complex assembly containing endogenous NCT are retained within the ER and that the incorporation of the fourth and last binding partner (PEN-2) also occurs on immature NCT, suggesting a complete assembly of the gamma-secretase complex within the ER.

Amyloid Precursor Protein Secretases↗
PubMed2006

Parallel assembly of dipolar columns composed of a stacked cyclic tri-beta-peptide.

A novel cyclic trimer of a beta-amino acid, trans-2-aminocyclohexylcarboxylic acid, was synthesized and its conformation and ability to form assemblies investigated. FT-IR and NMR measurements and computational calculations showed that this cyclic tri-beta-peptide has a C3-symmetric conformation with trans amide groups. A notable feature of the conformation is a vertical and parallel orientation of the three amide groups to the cyclic skeleton. The cyclic tri-beta-peptide was crystallized from a solution in trifluoroacetic acid-methanol (or trifluoroacetic acid-water) to yield a rod-shaped molecular assembly, as observed by TEM. The electron crystallography of the rod-shaped assembly both in suspension and in ultrathin cross-section revealed that the cyclic tri-beta-peptides were stacked up to form molecular columns, and that a two-fold screw symmetry operation along the column direction was present in the unit cell, which contained two cyclic tri-beta-peptides. This indicates that all the amide groups are oriented in the same direction. Since any two molecular columns are staggered by a quarter of a c-axis length and aligned parallel to each other, the dipole moments of the columns are aligned to enhance the strength additively in the whole assembly.

Crystallization↗
PubMed1993

Mechanisms of collagen trimer formation. Construction and expression of a recombinant minigene in HeLa cells reveals a direct effect of prolyl hydroxylation on chain assembly of type XII collagen.

Collagen types IX, XII, and XIV are characterized by the presence of a highly conserved region comprising the most C-terminal triple helical domain (COL1, approximately 100 residues/chain) and 2 cysteines separated by 4 amino acid residues at the junction between this COL1 domain and the C-terminal non-triple helical domain (NC1). In order to better understand the functions of this conserved domain, we have constructed a recombinant minigene, comprising the sequence coding for an unrelated signal peptide and for the COL1 and NC1 domains of type XII collagen. This construct was placed under the control of the cytomegalovirus promoter and transfected into HeLa cells. The cells expressed the transfected minigene and the secreted chain, called alpha 1 (mini XII), could be detected by immunotransfer with an anti-peptide antibody recognizing an epitope found in the NC1 domain. Under conditions preventing the hydroxylation of prolyl residues (absence of ascorbate or presence of alpha alpha'-dipyridyl), interchain disulfide bridges did not form, while in the presence of ascorbate, disulfide-bonded (alpha 1 (mini XII))3 molecules were secreted. The collagenous nature and triple helical conformation of the trimeric molecule were ascertained by the differential resistances of the COL1 and NC1 domains to trypsin and collagenase digestions, respectively. Our data demonstrate that the NC1 and COL1 domains of type XII collagen contain the information necessary for trimer formation and that, contrary to the fibrillar collagen types, posttranslational modification of the triple helical domain is essential for assembly and disulfide bonding of the chains.

Animals↗