The role of MutS oligomers on Pseudomonas aeruginosa mismatch repair system activity

The Escherichia coli DNA Mismatch Repair (MMR) protein MutS exist as dimers and tetramers in solution, and the identification of its functional oligomeric state has been matter of extensive study. In the present work, we have analyzed the oligomerization state of MutS from Pseudomonas aeruginosa a bacterial species devoid of Dam methylation and MutH homologue. By analyzing native MutS and different mutated versions of the protein, we determined that P. aeruginosa MutS is mainly tetrameric in solution and that its oligomerization capacity is conducted as in E. coli, by the C-terminal region of the protein. The analysis of mismatch oligonucleotide binding activity showed that wild-type MutS binds to DNA as tetramer. The DNA binding activity decreased when the C-terminal region was deleted (MutSDelta798) or when a full-length MutS with tetramerization defects (MutSR842E) was tested. The ATPase activity of MutSDelta798 was similar to MutSR842E and diminished respect to the wild-type protein. Experiments carried out on a P. aeruginosa mutS strain to test the proficiency of different oligomeric versions of MutS to function in vivo showed that MutSDelta798 is not functional and that full-length dimeric version MutSR842E, is not capable of completely restoring the MMR activity of the mutant strain. Additional experiments carried out in conditions of high mutation rate induced by the base analogue 2-AP confirm that the dimeric version of MutS is not as efficient as the tetrameric wild-type protein to prevent mutations. Therefore, it is concluded that although dimeric MutS is sufficient for MMR activity, optimal activity is obtained with the tetrameric version of the protein and therefore it should be considered as the active form of MutS in P. aeruginosa.     

The activity of barley NADPH-dependent thioredoxin reductase C is independent of the oligomeric state of the protein: tetrameric structure determined by cryo-electron microscopy

Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H(2)O(2). These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hordeum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.     

Differential accessibility of the carbohydrate moieties of Cls-Clr-Clr-Cls, the catalytic subunit of human Cl

The catalytic subunit of human Cl, Cls-Clr-Clr-Cls, is a Ca2+-dependent tetrameric association of two serine proteases, Clr and Cls, which are glycoproteins containing asparagine-linked carbohydrates. With a view to investigate the accessibility and the possible functional role of these carbohydrates, the isolated proteases and their Ca2+-dependent complexes were submitted to deglycosylation by peptide:N-glycosidase F, an endoglycosidase that specifically hydrolyzes all classes of N-linked glycans. Treatment of isolated Clr and Cls led to the removal of the carbohydrate moieties attached to their N-terminal alpha region, whereas those located in the C-terminal gamma-B catalytic domains were resistant to hydrolysis. Formation of the Ca2+-dependent Cls-Cls dimer and Cls-Clr-Clr-Cls tetramer induced specific protection of the single carbohydrate attached to the alpha region of Cls and of one of the two carbohydrates located in the corresponding region of Clr. Sequence studies indicated that the carbohydrates protected upon homologous (Cls-Cls) or heterologous (Clr-Cls) interactions are attached to asparagine residues 159 of Cls and 204 of Clr, at the C-terminal end of the EGF-like domain of both proteases. These data bring further evidence that Ca2+-dependent interactions between Clr and Cls are mediated by their N-terminal alpha regions and strongly suggest that, inside these regions, the EGF-like domains play an essential role in these interactions.     

Structural evolution of C-terminal domains in the p53 family

The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal beta-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.     

Autonomous Tetramerization Domains in the Glycan-binding Receptors DC-SIGN and DC-SIGNR

Multivalent binding of glycans on pathogens and on mammalian cells by the receptors DC-SIGN (CD209) and DC-SIGNR (L-SIGN, CD299) is dependent on correct disposition of the C-type carbohydrate-recognition domains projected at the C-terminal ends of necks at the cell surface. In the work reported here, neck domains of DC-SIGN and DC-SIGNR expressed in isolation are shown to form tetramers in the absence of the CRDs. Stability analysis indicates that interactions between the neck domains account fully for the stability of the tetrameric extracellular portions of the receptors. The neck domains are approximately 40% α-helical based on circular dichroism analysis. However, in contrast to other glycan-binding receptors in which fully helical neck regions are intimately associated with C-terminal C-type CRDs, the neck domains in DC-SIGN and DC-SIGNR act as autonomous tetramerization domains and the neck domains and CRDs are organized independently. Neck domains from polymorphic forms of DC-SIGNR that lack some of the repeat sequences show modestly reduced stability, but differences near the C-terminal end of the neck domains lead to significantly enhanced stability of DC-SIGNR tetramers compared to DC-SIGN.     

Amino acid and nucleotide sequence homologies among E. coli RNA polymerase core enzyme subunits, DNA primase, elongation factor Tu, F1-ATPase alpha, ribosomal protein L3, DNA polymerase I, T7 phage DNA polymerase, and MS2 phage RNA replicase beta subunit

The 330 residue-long N-terminal domains (NTDs) of beta and beta' subunits of the Escherichia coli RNA polymerase (RPase) core enzyme were found to be significantly homologous to the entire length of its alpha subunit. The C-terminal domains (CTDs) of the RPase beta subunit and DNA primase (dnaG protein) were not only strongly homologous to each other but also considerably homologous to the RPase alpha, suggesting that an alpha subunit-like enzyme must have been commonly ancestral to core enzyme subunits and primase. The N-terminal region (NTR) of RPase alpha was also found to show significant homologies with NTRs of the E. coli EF-Tu and F1-ATPase alpha subunit, and a possible weak homology with ribosomal protein L3. A most important finding was that the C-terminal regions (CTRs) of DNA polymerase (DPase) I, T7 phage DPase and MS2 phage RNA replicase beta subunit are closely homologous with one another. These CTRs showed considerable homologies to RPase alpha NTD and RPase beta CTD. These conclusions are based on statistical evaluations of homologies in base and/or amino acid sequence alignments.     

Oligomerization of Chicken Acetylcholinesterase Does Not Require Intersubunit Disulfide Bonds

Abstract: Acetylcholinesterase (AChE) is secreted from muscle and nerve cells and associates as multimers through intermolecular covalent and noncovalent bonds. The amino acid sequence of the C-terminus is thought to play an important role in these interactions. We generated mutants in the C-terminus of the catalytic T-subunit of chicken AChE to determine the importance of this region to oligomerization and to the amphipathic character of the protein. Wild-type recombinant chicken AChE secreted from human embryonic kidney 293 cells was assembled into dimers and tetramers exclusively. Mutants lacking the C-terminal Cys⁷⁶⁴, the only cysteine involved in interchain disulfide bonds, showed lower but significant levels of the secreted dimeric and tetrameric forms. A truncated mutant, lacking the C-terminal 39 amino acids, exhibited a severe decrease in content of the multimeric forms, yet small amounts of the dimer were detectable. The amphipathic character was dependent on the state of oligomerization. When analyzed by sucrose gradients, the sedimentation of tetramers was not affected by detergent, but monomers and dimers sedimented more slowly in the presence of detergent. Most of the recombinant wild-type enzyme, shown to be dimeric and tetrameric by sedimentation analysis, was monomeric when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that much of the secreted oligomeric AChE was not disulfide bonded. These data suggest that disulfide bonding of Cys⁷⁶⁴ is not required for the catalytic subunit of chicken AChE to form oligomers and that regions outside of the C-terminus contribute to the hydrophobic interactions that are important for stabilizing the oligomeric forms.     

tRNA glycylation system from Thermus thermophilus. tRNAGly identity and functional interrelation with the glycylation systems from other phylae.

The systems of tRNA glycylation belong to the most complex aminoacylation systems since neither the oligomeric structure of glycyl-tRNA synthetases (GlyRS) nor the discriminator bases in tRNAGly are conserved in the phylae. To better understand the structure-function relationship in glycylation systems of various origins and the functional peculiarities related to their structural divergences, the elements in tRNA conferring its glycine identity in Thermus thermophilus were characterized and compared to those of other systems. Thermophilic identity is conferred by the G1-C72, C2-G71, G3-C70, and C50-G64 pairs together with the G10, U16, C35, and C36 single residues. In contrast to most other aminoacylation systems, the discriminator base is not directly involved in identity. Transplantation of these elements in tRNAAsp and tRNAPhe converts specificity toward glycine albeit conservation of nucleotide 73. Analysis of the functional interrelation of the identity elements shows coupling in synthetase recognition of the elements from anticodon and G10 whereas those from acceptor arm are recognized independently. Despite nondirect implication in identity, the discriminator base contributes cooperatively with C36 in specificity of glycylation. The link between the structural heterogeneity and the functional divergence of the glycylation systems and the phylogenic interrelation of these systems were approached by comparing the ability of GlyRSs of various phylae to glycylate heterologous tRNAGly. Dimeric GlyRSs from mammalian and archaebacteria acylate efficiently only eukaryotic and archaebacterial tRNAGly with a discriminatory A73, whereas tetrameric Escherichia coli GlyRS acylates only eubacterial tRNAGly with a discriminatory U73. In contrast, dimeric yeast GlyRS acylates efficiently both eukaryotic and archaebacterial tRNAGly as well as peculiar prokaryotic isoacceptors. Species specificity is lost with the dimeric GlyRS from Thermus thermophilus that acylates efficiently eubacterial, archaebacterial, and eukaryotic tRNAGly. These features are discussed in the context of the evolution of the glycylation systems and the phylogenic interrelation of the organisms.     

PDZ支架蛋白 PDZK1通过PDZ1与PDZ3结构域与磷脂酶Cβ2羧基末端PDZ结合模块相互作用

磷脂酶C β (PLCβ)在G蛋白偶联受体 (GPCR)介导的细胞信号转导中发挥重要作用. 通过水解磷脂酰肌醇4,5二磷酸 (PIP2),磷脂酶C β可以产生3种重要的第二信使分子：二乙酰甘油 (DAG)、三磷酸肌醇 (IP3)和质子. 在果蝇中,磷脂酶C β通过它的羧基末端盘状同源区域结合模块 (PBM)与盘状同源区域 (PDZ)支架蛋白-失活无后电位D蛋白 (INAD)相互作用,从而调节果蝇的光信号传导 . 在哺乳动物中,磷脂酶C β家族有4个亚型,每1个亚型的羧基末端都有1个典型的盘状同源区域结合模块. 这一结构特点提示我们,磷脂酶C β可能通过其羧基末端的盘状同源区域结合模块与盘状同源区域支架蛋白相互作用,进而调节它们自身的细胞定位和功能. 然而,目前仍对哺乳动物磷脂酶C β家族的盘状同源区域结合蛋白知之甚少. 本文运用分析型凝胶过滤和等温滴定量热技术,系统地研究了不同磷脂酶Cβ亚型的羧基末端盘状同源区域结合模块与不同盘状同源区域蛋白质的结合. 结果表明,磷脂酶Cβ2的羧基末端盘状同源区域结合模块,可以特异地与含有4个盘状同源区域的支架蛋白-盘状同源区域蛋白1 (PDZK1）以2∶1的方式相互结合. 进一步的测定显示,磷脂酶C β2羧基末端盘状同源区域结合模块在盘状同源区域蛋白1上的结合位点为第1和第3个盘状同源区域,而它们与磷脂酶Cβ2的解离常数分别为11.8±3.4 μmol/L 和33.3±8.7 μmol/L.     

Activity of the tetramer and octamer of glutamine synthetase isoforms during primary leaf ontogeny of sugar beet (Beta vulgaris L.)

In extracts from the primary leaf blade of sugar beet (Beta vulgaris L.) we separated a chloroplastic isoform (GS 2) of glutamine synthetase (GS, EC 6.3.1.2) and one or two (depending on leaf age) cytosolic isoforms (GS 1a and GS 1b). The latter were prominent in the early (GS 1a) and late stages of leaf ontogeny (GS 1a and GS 1b), whereas during leaf maturation GS 2 was the predominantly active GS isoform. The GS 1 isoforms were active exclusively in the octameric state although tetrameric GS 1 protein was detected immunologically. Their activity stayed at a relatively constant level during leaf ontogeny; an increase was observed only in the senescent leaf. The activity of GS 2, however, changed drastically during primary leaf ontogeny and was modulated by changes in the oligomeric state of the active enzyme. In the early and late stages of leaf ontogeny when GS 2 activity was low (lower than that of the GS 1 isoforms), GS 2 was active only in the octameric state. In the maturing leaf, when GS 2 activity had reached its maximum level (much higher than that of the GS 1 isoforms), 80 of total GS 2 activity was due the activity of the tetrameric form of the enzyme and 20 was due to octameric GS 2. Tetrameric GS 2 was a hetero-tetramer and thus not the unspecific dissociation product of homo-octameric GS 2. In addition, GS 2 activity was modulated by an activation/inactivation of the tetrameric GS 2 protein. Due to an activation of the GS 2 tetramer, the activity of tetrameric GS 2 increased during leaf maturation from zero level 23-fold compared with that of GS 1a and 18-fold compared with that of GS 1b. Possible activators of tetrameric GS 2 are thiol-reactive substances. During leaf senescence, GS 2 activity decreased to zero; this decrease was due to an inactivation of the tetrameric GS 2 protein probably caused by oxidation.Abbreviations FLL final lamina length - FPLC fast protein liquid chromatography - GS glutamine synthetase - GHA -glutamyl hydroxamate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Dr. Roger Wallsgrove's (Rothamsted Experimental Station, Harpenden, UK) generous gift of GS antiserum is greatly appreciated.     

Structural studies of MFE-1: the 1.9 A crystal structure of the dehydrogenase part of rat peroxisomal MFE-1

The 1.9 A structure of the C-terminal dehydrogenase part of the rat peroxisomal monomeric multifunctional enzyme type 1 (MFE-1) has been determined. In this construct (residues 260-722 and referred to as MFE1-DH) the N-terminal hydratase part of MFE-1 has been deleted. The structure of MFE1-DH shows that it consists of an N-terminal helix, followed by a Rossmann-fold domain (domain C), followed by two tightly associated helical domains (domains D and E), which have similar topology. The structure of MFE1-DH is compared with the two known homologous structures: human mitochondrial 3-hydroxyacyl-CoA dehydrogenase (HAD; sequence identity is 33%) (which is dimeric and monofunctional) and with the dimeric multifunctional alpha-chain (alphaFOM; sequence identity is 28%) of the bacterial fatty acid beta-oxidation alpha2beta2-multienzyme complex. Like MFE-1, alphaFOM has an N-terminal hydratase part and a C-terminal dehydrogenase part, and the structure comparisons show that the N-terminal helix of MFE1-DH corresponds to the alphaFOM linker helix, located between its hydratase and dehydrogenase part. It is also shown that this helix corresponds to the C-terminal helix-10 of the hydratase/isomerase superfamily, suggesting that functionally it belongs to the N-terminal hydratase part of MFE-1.     

Structural basis for phosphotyrosine recognition by the Src homology-2 domains of the adapter proteins SH2-B and APS

SH2-B, APS, and Lnk constitute a family of adapter proteins that modulate signaling by protein tyrosine kinases. These adapters contain an N-terminal dimerization region, a pleckstrin homology domain, and a C-terminal Src homology-2 (SH2) domain. SH2-B is recruited via its SH2 domain to various protein tyrosine kinases, including Janus kinase-2 (Jak2) and the insulin receptor. Here, we present the crystal structure at 2.35 A resolution of the SH2 domain of SH2-B in complex with a phosphopeptide representing the SH2-B recruitment site in Jak2 (pTyr813). The structure reveals a canonical SH2 domain-phosphopeptide binding mode, but with specific recognition of a glutamate at the +1 position relative to phosphotyrosine, in addition to recognition of a hydrophobic residue at the +3 position. Biochemical studies of SH2-B and APS demonstrate that, although the SH2 domains of these two adapter proteins share 79% sequence identity, the SH2-B SH2 domain binds preferentially to Jak2, whereas the APS SH2 domain has higher affinity for the insulin receptor. This differential specificity is attributable to the difference in the oligomeric states of the two SH2 domains: monomeric for SH2-B and dimeric for APS.     

A hydrophobic segment within the C-terminal domain is essential for both client-binding and dimer formation of the HSP90-family molecular chaperone.

The alpha isoform of human 90-kDa heat shock protein (HSP90alpha) is composed of three domains: the N-terminal (residues 1-400); middle (residues 401-615) and C-terminal (residues 621-732). The middle domain is simultaneously associated with the N- and C-terminal domains, and the interaction with the latter mediates the dimeric configuration of HSP90. Besides one in the N-terminal domain, an additional client-binding site exists in the C-terminal domain of HSP90. The aim of the present study is to elucidate the regions within the C-terminal domain responsible for the bindings to the middle domain and to a client protein, and to define the relationship between the two functions. A bacterial two-hybrid system revealed that residues 650-697 of HSP90alpha were essential for the binding to the middle domain. An almost identical region (residues 657-720) was required for the suppression of heat-induced aggregation of citrate synthase, a model client protein. Replacement of either Leu665-Leu666 or Leu671-Leu672 to Ser-Ser within the hydrophobic segment (residues 662-678) of the C-terminal domain caused the loss of bindings to both the middle domain and the client protein. The interaction between the middle and C-terminal domains was also found in human 94-kDa glucose-regulated protein. Moreover, Escherichia coli HtpG, a bacterial HSP90 homologue, formed heterodimeric complexes with HSP90alpha and the 94-kDa glucose-regulated protein through their middle-C-terminal domains. Taken together, it is concluded that the identical region including the hydrophobic segment of the C-terminal domain is essential for both the client binding and dimer formation of the HSP90-family molecular chaperone and that the dimeric configuration appears to be similar in the HSP90-family proteins.     

Domain structures and molecular evolution of class I and class II major histocompatibility gene complex (MHC) products deduced from amino acid and nucleotide sequence homologies

Domain structures of class I and class II MHC products were analyzed from a viewpoint of amino acid and nucleotide sequence homologies. Alignment statistics revealed that class I (transplantation) antigen H chains consist of four mutually homologous domains, and that class II (HLA-DR) antigen beta and alpha chains are both composed of three mutually homologous ones. The N-terminal three and two domains of class I and class II (both beta and alpha) gene products, respectively, all of which being approximately 90 residues long, were concluded to be homologous to beta2-microglobulin (beta2M). The membrane-embedded C-terminal shorter domains of these MHC products were also found to be homologous to one another and to the third domain of class I H chains. Class I H chains were found to be more closely related to class II alpha chains than to class II beta chains. Based on these findings, an exon duplication history from a common ancestral gene encoding a beta2M-like primodial protein of one-domain-length up to the contemporary MHC products was proposed.     

In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities.

Plasmid lambdadv1, which is in a dimeric form, was converted to a linear monomer duplex by the action of EcoRI restriction endonuclease that incises at a unique site in this plasmid genome. The resulting products were then joined by Escherichia coli DNA ligase to produce molecules with various oligomeric forms, and from these monomeric, dimeric, or trimeric circular molecules were purified. By transformation of cells with these DNAs, clones were obtained that carried lambdadv1 in a monomeric or dimeric form. The former type of clones have not been generated in vivo, except for one in a different host strain, and carriers of timeric or tetrameric lambdadv1's have not been obtained so far. It was observed that a considerable fraction of these oligomeric circular DNAs were converted to lower oligomers (e.g., from trimer to dimer) during transformation. The characteristics of the monomeric lambdadv1 carriers obtained were compared with those of dimeric lambdadv1 carriers. The stabilities of the plasmids of the two forms were the same. However, the monomeric plasmid carriers were less tolerant to lambdavir phage infection and perpetuated about 30% less plasmid genomes in monomer units. Furthermore, dimeric plasmid carriers appeared spontaneously and accumulated in cultures of the monomeric lambdadv1 carriers.     

The link between restriction endonuclease fidelity and oligomeric state: A study with Bse634I

Type II restriction endonucleases (REases) exist in multiple oligomeric forms. The tetrameric REases have two DNA binding interfaces and must synapse two recognition sites to achieve cleavage. It was hypothesised that binding of two recognition sites by tetrameric enzymes contributes to their fidelity. Here, we experimentally determined the fidelity for Bse634I REase in different oligomeric states. Surprisingly, we find that tetramerisation does not increase REase fidelity in comparison to the dimeric variant. Instead, an inherent ability to act concertedly at two sites provides tetrameric REase with a safety-catch to prevent host DNA cleavage if a single unmodified site becomes available.     

Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).     

Genetic Design of Stable Metal-Binding Biomolecules,Oligomeric Metallothioneins

Metallothionein (MT) is a suitable model for investigating molecular interactions relating to the handling of metals in cells. However, the production of functional MT proteins in microorganisms has been limited because of the instability of MT—the thiol group of cysteine is easily oxidized and proteolysis occurs. To increase the binding ability and to stabilize MT, we designed genes for dimeric and tetrameric MT and the genes were successfully overexpressed in Escherichia coli to generate functional oligomeric MTs. A human MT-II (hMT-II) synthesized with prokaryotic codons, a linker encoding a glycine tripeptide, and Met-deficient hMT-II was ligated to create a dimeric MT, from which a tetrameric MT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. Cells of E. coli carrying the oligomeric MT genes showed resistance toward Zn and Cd toxicity. The oligomeric proteins formed inclusion bodies, which were dissolved with dithiothreitol, and the purified apo-MTs were reconstituted with Cd or Zn ions under reducing conditions. The dimeric and tetrameric MT proteins exhibited both Cd and Zn binding activities that were, respectively, two and four times higher than those of the hMT-II monomer protein. These stable oligomeric MTs have potential as a biomaterial for uses such as detoxification and bioremediation for heavy metals.     

Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase beta.

Interaction of flavin mononucleotide (FMN) with dimeric and tetrameric forms of rabbit muscle glycogen phosphorylase beta has been studied under the conditions when allosteric activator binding sites are saturated by AMP (1 mM AMP; pH 6.8; 17 degrees C). Simultaneous use of schlieren optical system and photoelectric scanning absorption optical system of analytical ultracentrifuge Spinco, model E, makes it possible to register the oligomeric state of the enzyme and calculate the degree of saturation of individual oligomeric enzyme forms by FMN. The apparent association constant for the equilibrium dimer in equilibrium with tetramer decreased with increasing FMN concentration. The microscopic dissociation constants for the complexes of dimeric and tetrameric forms of glycogen phosphorylase beta with FMN have been found to be equal to 10 and 79 microM, respectively.