Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation.

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Structure analysis of short peptides by analytical ultracentrifugation: Review.

Short peptides are potential drug candidates for pharmaceutical and biotech industries. Short peptides are natural ligands for numerous G-protein coupled receptors (GPCR) and hence constitute a large number of drug candidates. Synthetic short peptides are also extensively developed as agonistic or antagonistic ligands that function in a similar manner to antibodies, soluble receptors and protein ligands. Characterization of the peptides in solution is often performed in the presence of organic solvents, which can presumably generate the structure bound to the target surface and also enhance the solubility of the peptides. Analytical ultracentrifugation (AUC) technique should provide information on the state of self-association of the peptide in solution. Its application for short peptides has been far less than the applications for proteins. We believe that AUC should be used to show the associated state of the peptides, as reviewed in this paper.

Purification, characterization and N-terminal sequences alignment of a mannose specific protein purified from Potca fish, Tetraodon patoca.

A protein was isolated and purified from the ventral portion of the Potca fish, Tetraodon patoca. The method was accomplished by gel filtration of crude protein extract on Sephadex G-50 followed by Ion exchange chromatography on DEAE-cellulose and finally by affinity chromatography on ConA-Sepharose matrix. The molecular weight of the protein, determined by the gel filtration and SDS-PAGE was about 82,000 and 80,000 respectively, but 42,000 and 38,000 were indicated by SDS-PAGE in the presence of 2-mercaptoethanol. The protein agglutinated rat red blood cells and in a haptein-inhibition test, the protein was inhibited specifically by the D-mannose and mannose containing saccharides. The protein is glycoprotein with neutral sugar content of about 0.35%. The purified protein also showed strong cytotoxic effects, which was performed by brine shrimp lethality bioassay and histopathological examinations. The N-terminal amino acid sequences of both the subunits of the protein were also identified and used a blast search on N-terminal amino acid sequences of the subunits revealed that the protein showed significant homology with the homologous proteins in database.

Single step purification, characterization and N-terminal sequences of a mannose specific lectin from mulberry seeds.

A mannose/glucose specific lectin have been isolated and purified from mulberry seeds by affinity chromatography on ConA Sepharose. The lectin is monomer in nature as judged by SDS-PAGE and its MW was estimated to be 22,000. The lectin is glycoprotein with neutral sugar content of 28.57%, and mannose and glucose were identified as carbohydrate. The lectin agglutinated rat red blood cells and in a hapten inhibition test, D: -mannose and D: -glucose was found to be inhibitor. The lectin also exhibited cytotoxic effect in brine shrimp lethality bioassay. The N-terminal sequences of the lectin upto 45-residues except the positions of 21, 39, 42 and 44 were identified. Sequence homology of the lectin is also discussed.

Structure and morphogenesis of bacteriophage T4.

Bacteriophage T4 is one of the most complex viruses. More than 40 different proteins form the mature virion, which consists of a protein shell encapsidating a 172-kbp double-stranded genomic DNA, a 'tail,' and fibers, attached to the distal end of the tail. The fibers and the tail carry the host cell recognition sensors and are required for attachment of the phage to the cell surface. The tail also serves as a channel for delivery of the phage DNA from the head into the host cell cytoplasm. The tail is attached to the unique 'portal' vertex of the head through which the phage DNA is packaged during head assembly. Similar to other phages, and also herpes viruses, the unique vertex is occupied by a dodecameric portal protein, which is involved in DNA packaging.

The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures.

Random mutagenesis coupled with screening of the active enzyme at a low temperature was applied to isolate cold-adapted mutants of a thermophilic enzyme. Four mutant enzymes with enhanced specific activities (up to 4.1-fold at 40 degrees C) at a moderate temperature were isolated from randomly mutated Thermus thermophilus 3-isopropylmalate dehydrogenase. Kinetic analysis revealed two types of cold-adapted mutants, i.e. k(cat)-improved and K(m)-improved types. The k(cat)-improved mutants showed less temperature-dependent catalytic properties, resulting in improvement of k(cat) (up to 7.5-fold at 40 degrees C) at lower temperatures with increased K(m) values mainly for NAD. The K(m)-improved enzyme showed higher affinities toward the substrate and the coenzyme without significant change in k(cat) at the temperatures investigated (30-70 degrees C). In k(cat)-improved mutants, replacement of a residue was found near the binding pocket for the adenine portion of NAD. Two of the mutants retained thermal stability indistinguishable from the wild-type enzyme. Extreme thermal stability of the thermophilic enzyme is not necessarily decreased to improve the catalytic function at lower temperatures. The present strategy provides a powerful tool for obtaining active mutant enzymes at lower temperatures. The results also indicate that it is possible to obtain cold-adapted mutant enzymes with high thermal stability.

Stoichiometry and inter-subunit interaction of the wedge initiation complex, gp10-gp11, of bacteriophage T4.

Association of gp10 and gp11 (gp=gene product) is the first step in the assembly pathway of the wedge part of the baseplate of bacteriophage T4. The gp10-gp11 complex constitutes the six tail pins at the corners of the baseplate hexagon on the distal side. The stoichiometry of the subunits, gp10 and gp11, of this complex was determined in combination with sedimentation equilibrium, Edman degradation of the complex and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of Edman degradation and SDS-PAGE, the molar ratio of gp10 and gp11 was approximately 1. On the other hand, the molecular weight of the purified gp10-gp11 complex was determined by sedimentation equilibrium to be 284000+/-7000, which is in good agreement with the expected value of 269840 if the stoichiometry is 3:3. Furthermore, comparison of the results in the presence and in the absence of reducing reagent, 2-mercaptoethanol (2-ME), in SDS-PAGE revealed that two molecules of gp10 in the complex formed a disulfide bond, while the third gp10 molecule does not participate in the disulfide bond formation.

Silk fibroin of Bombyx mori is secreted, assembling a high molecular mass elementary unit consisting of H-chain, L-chain, and P25, with a 6:6:1 molar ratio.

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.

Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches.

Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure. In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out. PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method. The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved. Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously. Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90.

Chimeric virus-like particle formation of adeno-associated virus.

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3. These capsid proteins have a common amino acid sequence, being expressed from different initiation codons on the same open reading frame. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but their functions are poorly understood. To investigate the role(s) of the capsid proteins in capsid formation, we used a baculovirus protein expression system to produce virus-like particles (VLPs). We found that varying the ratios of VP2 and VP3 did not affect VLP formation. Further, their physical properties were equivalent to those of empty wild-type particles. The function of VP3 was studied further by fusing a peptide tag, FLAG, to its N-terminus. This chimeric viral protein, in combination with VP2, could assemble into VLPs, indicating that the chimerism of VP3 did not affect VLP formation. Although the monomeric native form of the FLAG-VP3 chimera could react with anti-FLAG antibody, VLP containing the chimeric VP3 could not, suggesting that the N-terminal region of VP3 is located inside the VLP. These observations indicate that it may be possible to utilize AAV VLP as vectors of a broad range of drugs since fusion of the VP3 N-terminus with defined molecules could impose distinct physical properties onto the internal environment of the VLP.

Nuclear transport of the major capsid protein is essential for adeno-associated virus capsid formation.

Adeno-associated virus capsids are composed of three proteins, VP1, VP2, and VP3. Although VP1 is necessary for viral infection, it is not essential for capsid formation. The other capsid proteins, VP2 and VP3, are sufficient for capsid formation, but the functional roles of each protein are still not well understood. By analyzing a series of deletion mutants of VP2, we identified a region necessary for nuclear transfer of VP2 and found that the efficiency of nuclear localization of the capsid proteins and the efficiency of virus-like particle (VLP) formation correlated well. To confirm the importance of the nuclear localization of the capsid proteins, we fused the nuclear localization signal of simian virus 40 large T antigen to VP3 protein. We show that this fusion protein could form VLP, indicating that the VP2-specific region located on the N-terminal side of the protein is not structurally required. This finding suggests that VP3 has sufficient information for VLP formation and that VP2 is necessary only for nuclear transfer of the capsid proteins.

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.

A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides.

A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.