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PubMed1991

Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function.

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.

Amino Acid Sequence↗
PubMed2002

Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly.

The protein regions in the S-layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self-assembly, were mapped by deletion analysis of His-tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32-271 carries the infor-mation for self-assembly of CbsA into a periodic structure. The lactobacillar S-layer proteins exhibit sequence variation in their assembly domain, but the border regions 30-34 and 269-274 in CbsA are conserved in valine-rich short sequences. Short deletions or substitutions at these regions affected the morphology of His-CbsA polymers, which varied from sheet-like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non-periodic aggregation. The self-assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross-linking study. The shorter peptides not forming a regular polymer were observed by the cross-linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross-linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His-CbsA peptides 1-274 and 31-287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1-274, 1-287, 28-287 and 31-287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin- and collagen-containing regions in chicken colon and ileum. The C-terminal peptides 251-410 and 288-410 bound to L. crispatus JCM 5810 cells from which the S-layer had been depleted by chemical extraction, whereas no binding was seen with the His-CbsA peptides 1-250 or 1-269 or to cells with an intact S-layer. The His-CbsA peptides 251-410 and 288-410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N-terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C-terminal domain binding to negatively charged cell wall components.

Bacterial Adhesion↗
PubMed2005

Neutral Cu4N12 and Ag4N12 metallacycles with a para-cyclophane framework assembled from copper(I) and silver(I) pyrazolates and pyridazine.

Trinuclear copper(I) and silver(I) pyrazolates {[3,5-(CF3)2Pz]M}3 (M = Cu and Ag) react with pyridazine to give neutral, tetranuclear metallacycles with a para-cyclophane core whereas benzo[c]cinnoline fails to break the cyclic pyrazolate trimers under similar conditions, and affords a metalla-propellane featuring both two- and three-coordinate metal sites.

Journal Article↗
PubMed1996

Assembly of the gigantic hemoglobin of the earthworm Lumbricus terrestris. Roles of subunit equilibria, non-globin linker chains, and valence of the heme iron.

The extracellular hemoglobin of the earthworm Lumbricus terrestris has four major kinds of O2-binding chains: a, b, and c (forming a disulfide-linked trimer), and chain d. Non-heme, non-globin structural chains, "linkers," are also present. Light-scattering techniques have been used to show that the ferrous CO-saturated abc trimer and chain d form an (abcd)4 complex of 285 kDa at neutral pH. Formation of the full-sized 4-MDa molecule requires the addition of linker chains in the proportion of two linkers per (abcd)4 and occurs much more rapidly in the presence of 10 mM calcium. This stoichiometry is supported not only by direct quantitative analysis of the intact hemoglobin but also by the fact that the addition of 50% of the proposed stoichiometric quantity of linkers results in the conversion of 50% of the (abcd)4 to full-sized molecules. Isolated CO-saturated abc trimers self-associate to (abc)2 and higher aggregates up to an apparent limit of (abc)10 approximately 550 kDa. The CO-saturated chain d forms dimers, (d)2, and tetramers, (d)4. Oxidation of the (abcd)4 complex with ferricyanide causes complete dissociation of chain d from the abc trimer, but addition of CN- maintains the (abcd)4 complex. Valence hybrids have also been studied. The ferrous CO-saturated abc trimer and met (ferric) chain d also associate to form (abcd)4, but the met abc trimer and ferrous CO-saturated chain d do not. Oxidation of the abc trimer and chain d to the ferric form causes the formation of a characteristic hemichrome spectrum with a maximum at 565 nm and a shoulder near 530 nm. These results show that interactions between the abc trimer and chain d are strongly dependent on the ligand and valence state of the heme iron. Light-scattering measurements reveal that oxidation of the intact Hb produces a significant drop in molecular mass from 4.1 to 3.6 MDa. Inclusion of CN- prevents this drop. These experiments indicate that oxidation causes the Hb to shed subunits. The observations provide an explanation for the wide variations in the molecular mass of L. terrestris Hb that have been observed previously.

Alcohol Dehydrogenase↗
PubMed2005

RNA nanotechnology: engineering, assembly and applications in detection, gene delivery and therapy.

Biological macromolecules including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. RNA is unique in nanoscale fabrication due to its amazing diversity of function and structure. RNA molecules can be designed and manipulated with a level of simplicity characteristic of DNA while possessing versatility in structure and function similar to that of proteins. RNA molecules typically contain a large variety of single stranded loops suitable for inter- and intra-molecular interaction. These loops can serve as mounting dovetails obviating the need for external linking dowels in fabrication and assembly. The self-assembly of nanoparticles from RNA involves cooperative interaction of individual RNA molecules that spontaneously assemble in a predefined manner to form a larger two- or three-dimensional structure. Within the realm of self-assembly there are two main categories, namely template and non-template. Template assembly involves interaction of RNA molecules under the influence of specific external sequence, forces, or spatial constraints such as RNA transcription, hybridization, replication, annealing, molding, or replicas. In contrast, non-template assembly involves formation of a larger structure by individual components without the influence of external forces. Examples of non-template assembly are ligation, chemical conjugation, covalent linkage, and loop/loop interaction of RNA, especially the formation of RNA multimeric complexes. The best characterized RNA multiplier and the first to be described in RNA nanotechnological application is the motor pRNA of bacteriophage phi29 which form dimers, trimers, and hexamers, via hand-in-hand interaction. phi29 pRNA can be redesigned to form a variety of structures and shapes including twins, tetramers, rods, triangles, and 3D arrays several microns in size via interaction of programmed helical regions and loops. 3D RNA array formation requires a defined nucleotide number for twisting and a palindromic sequence. Such arrays are unusually stable and resistant to a wide range of temperatures, salt concentrations, and pH. Both the therapeutic siRNA or ribozyme and a receptor-binding RNA aptamer or other ligands have been engineered into individual pRNAs. Individual chimeric RNA building blocks harboring siRNA or other therapeutic molecules have been fabricated subsequently into a trimer through hand-in-hand interaction of the engineered right and left interlocking RNA loops. The incubation of these particles containing the receptor-binding aptamer or other ligands results in the binding and co-entry of trivalent therapeutic particles into cells. Such particles were subsequently shown to modulate the apoptosis of cancer cells in both cell cultures and animal trials. The use of such antigen-free 20-40 nm particles holds promise for the repeated long-term treatment of chronic diseases. Other potentially useful RNA molecules that form multimers include HIV RNA that contain kissing loop to form dimers, tecto-RNA that forms a "jigsaw puzzle," and the Drosophila bicoid mRNA that forms multimers via "hand-by-arm" interactions. Applications of RNA molecules involving replication, molding, embossing, and other related techniques, have recently been described that allow the utilization of a variety of materials to enhance diversity and resolution of nanomaterials. It should eventually be possible to adapt RNA to facilitate construction of ordered, patterned, or pre-programmed arrays or superstructures. Given the potential for 3D fabrication, the chance to produce reversible self-assembly, and the ability of self-repair, editing and replication, RNA self-assembly will play an increasingly significant role in integrated biological nanofabrication. A random 100-nucleotide RNA library may exist in 1.6 x 10(60) varieties with multifarious structure to serve as a vital system for efficient fabrication, with a complexity and diversity far exceeding that of any current nanoscale system. This review covers the basic concepts of RNA structure and function, certain methods for the study of RNA structure, the approaches for engineering or fabricating RNA into nanoparticles or arrays, and special features of RNA molecules that form multimers. The most recent development in exploration of RNA nanoparticles for pathogen detection, drug/gene delivery, and therapeutic application is also introduced in this review.

Gene Transfer Techniques↗
PubMed1996

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.

Allosteric Site↗
PubMed2005

Differences in pairing and cluster formation of T cell receptor alpha- and beta-chains in T cell clones and fusion hybridomas.

The questions of T cell receptor (TCR) clustering and preferential pairing of TCR alpha- and beta-chains are discussed controversially. We here describe the rare case of a non-pairing TCR alpha-TCR beta combination detected in the murine T cell hybridoma Hy-E6. Of its two TCR alpha-chains (Valpha3.2, Vbeta17) and one Vbeta16-chain only the Valpha17/Vbeta16 TCR is exposed on the surface, despite intracellular expression of Valpha3.2 protein. The lack of Valpha3.2/Vbeta16 pairing was confirmed by TCR transfections. Surprisingly, however, the parental T cell clone CTL-E6 expressed both alpha-chains on its plasma membrane. Different size distribution of TCR clusters in CTL-E6 versus Hy-E6 and transfectants as determined by Blue-Native gel electrophoresis indicated differences in the supra-molecular TCR assembly as one possible reason for this phenomenon. Our data further reveal that the nominal specificity of CTL-E6 for the fully agonistic trinitrophenyl (TNP) modified peptide M4L-TNP was specifically mediated by the trimeric Valpha3.2/Valpha17/Vbeta16 TCR of CTL-E6. In contrast, the Valpha17/Vbeta16 combination in Hy-E6 only conferred specificity for the cross-reactive partial agonist O4-TNP. Both specificities are H-2Kb-restricted and, hence, appear to be positively selected. The differences in TCR clustering in CTL and hybridoma might indicate differences in the reception and transmission of TCR-signals between these two cell types.

Amino Acid Sequence↗
PubMed2005

Stability of the homopentameric B subunits of shiga toxins 1 and 2 in solution and the gas phase as revealed by nanoelectrospray fourier transform ion cyclotron resonance mass spectrometry.

The assembly of the B subunits of Shiga toxins (Stx) 1 and 2 and the influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on it are investigated using temperature-controlled nanoflow electrospray (nano-ES) ionization and Fourier-transform ion cyclotron resonance mass spectrometry. Despite the similar higher order structure predicted by X-ray crystallography analysis, the B(5) homopentamers of Stx1 and Stx2 exhibit differences in stability under the solution conditions investigated. At solution temperatures ranging from 0 to 60 degrees C and subunit concentrations ranging from 5 to 85 microM, the Stx1 B subunit exists almost entirely as the homopentamer in aqueous solutions, independent of the ionic strength. In contrast, the degree of assembly of Stx2 B subunit is strongly dependent on temperature, subunit concentration, and ionic strength. At subunit concentrations of more than 50 microM, the Stx2 B subunit exists predominantly as a pentamer, although smaller multimers (dimer, trimer, and tetramer) are also evident. At lower concentrations, the Stx2 B subunit exists predominantly as monomer and dimer. The relative abundance of multimeric species of the Stx2 B subunit was insensitive to the ion source conditions, suggesting that gas-phase dissociation of the pentamer ions in the source does not influence the mass spectrum. Blackbody infrared radiative dissociation of the protonated B(5) ions of Stx2 at the +12 and +13 charge states proceeds, at reaction temperatures of 120 to 180 degrees C, predominantly by the ejection of a single subunit from the complex. Dissociation into dimer and trimer ions constitutes a minor pathway. It follows that the dimer and trimer ions and, likely, the monomer ions observed in the nano-ES mass spectra of Stx2 B subunit originated in solution and not from gas-phase reactions. It is concluded that, under the solution conditions investigated, the homopentamer of Stx2 B subunit is thermodynamically less stable than that of Stx1 B subunit. Arrhenius activation parameters determined for the protonated Stx2 B(5) ions at the +12 and +13 charge states were compared with values reported for the corresponding B(5) ions of Stx1 B subunit. In contrast to the differential stability of the Stx1 and Stx2 B pentamers in solution, the dissociation activation energies (E(a)) determined for the gaseous complexes are indistinguishable at a given charge state. The similarity in the E(a) values suggests that the protonated pentamer ions of both toxins are stabilized by similar intersubunit interactions in the gas phase, a result that is in agreement with the X-ray crystal structures of the holotoxins.

Cyclotrons↗
PubMed2001

Supramolecular daisy chains.

Two series of self-complementary daisy chain monomers, in which a secondary ammonium ion-containing arm is grafted onto a macrocycle with either a [24]- or [25]crown-8 constitution, have been synthesized. In the solid- and 'gas'-phases, the parent [24]crown-8-based monomer forms dimeric superstructures, as revealed by X-ray crystallography and mass spectrometry, respectively. Elucidation of the complicated solution-phase behavior of this compound was facilitated by the synthesis and study of both deuterated, and fluorinated, analogues. These investigations revealed that the cyclic dimeric superstructure also dominates in solution, except when extremes of either concentration (low), temperature (high), or solvent polarity (highly polar, e.g., dimethyl sulfoxide) are employed. Whereas, upon aggregation, the [24]crown-8-based daisy chain monomers have the capacity to form stereoisomeric superstructures further complicating the study of this series of compounds. The assembly of [25]crown-8-based monomers gives only achiral superstructures. The weaker association exhibited between secondary dialkylammonium ions and crown ethers with a [25]crown-8 constitution, however, resulted in limited oligomerization--only dimeric and trimeric superstructures were formed at experimentally attainable concentrations--of [25]crown-8-based daisy chain monomers.

Journal Article↗
PubMed1997

A novel structural model for regulation of clathrin function.

The distinctive triskelion shape of clathrin allows assembly into polyhedral lattices during the process of clathrin-coated vesicle formation. We have used random and site-directed mutagenesis of the yeast clathrin heavy chain gene (CHC1) to characterize regions which determine Chc trimerization and binding to the clathrin light chain (Clc) subunit. Analysis of the mutants indicates that mutations in the trimerization domain at the triskelion vertex, as well as mutations in the adjacent leg domain, frequently influence Clc binding. Strikingly, one mutation in the trimerization domain enhances the association of Clc with Chc. Additional mutations in the trimerization domain, in combination with mutations in the adjacent leg domain, exhibit severe defects in Clc binding while maintaining near normal trimerization properties. The position of these trimerization domain mutations on one face of a putative alpha-helix defines a region on the trimer surface that interacts directly with Clc. These results suggest that Clc extends into the Chc trimerization domain from the adjacent leg, thereby bridging the two domains. On the basis of this conclusion, we propose a new model for the organization of the triskelion vertex which provides a structural basis for regulatory effects of Clc on clathrin function.

Amino Acid Sequence↗
PubMed1992

Control of translational repression by protein-protein interactions.

The coat protein of the RNA bacteriophage MS2 is a translational repressor and interacts with a specific RNA stem-loop to inhibit translation of the viral replicase gene. As part of an effort to dissect genetically its RNA binding function, mutations were identified in the coat protein sequence that suppress mutational defects in the translational operator. Each of the mutants displayed a super-repressor phenotype, repressing translation from the wild-type and a variety of mutant operators better than did the wild-type coat protein. At least one mutant probably binds RNA more tightly than wild-type. The other mutants, however, were defective for assembly of virus-like particles, and self-associated predominantly as dimers. It is proposed that this assembly defect accounts for their super-repressor characteristics, since failure to assemble into virus-like particles elevates the effective concentration of repressor dimers. This hypothesis is supported by the observation that deletion of thirteen amino acids known to be important for assembly of dimers into capsids also resulted in the same assembly defect and in super-repressor activity. A second class of assembly defects is also described. Deletion of two amino acids from the C-terminus of coat protein resulted in failure to form capsids, most of the coat protein having the apparent molecular weight expected of trimers. This mutant (dl-8) was completely defective for repressor activity, probably because of an inability to form dimers. These results point out the inter-dependence of the structural and regulatory functions of coat protein.

Amino Acid Sequence↗
PubMed1996

Structure of mouse interferon stimulated gene factor 3 gamma (ISGF3 gamma/p48) cDNA and chromosomal localization of the gene.

Interferon stimulated gene factor 3 (ISGF3) is a trimeric transcription factor activated on treatment of cells with interferon-alpha and beta (type I IFNs). Upon stimulation, the regulatory subunits, p84/91 and p113, present in the cytoplasm are phosphorylated at specific tyrosine residues and assemble with the DNA binding subunit, ISGF3 gamma, into the active ISGF3 in the nucleus. Thus, ISGF3 plays a primary role in the transmission of a signal from the cell surface to the nucleus. In this report, we describe the cloning of a mouse cDNA encoding a polypeptide homologous to human ISGF3 gamma. Comparison of the deduced amino acid sequences revealed the middle region was significantly different between mouse and man. The mouse cDNA was shown to encode a functional ISGF3 subunit by means of an in vitro reconstitution assay. Furthermore, the locus of the ISGF3 gamma gene, designated as Isgf3g, was mapped to distal mouse chromosome 14 by linkage analysis using an intersubspecific backcross typing panel.

Amino Acid Sequence↗
PubMed2006

The fellowship of the rings: distinct pools of proliferating cell nuclear antigen trimer at work.

The proliferating cell nuclear antigen (PCNA) is a homotrimeric ring-shaped protein that, by encircling DNA, may function as a sliding platform for proteins participating in various DNA transactions. PCNA plays a fundamental role in DNA replication and repair, but also in postreplicative events, like DNA methylation, chromatin assembly and remodeling, sister chromatid cohesion, and coordinates these activities with cell cycle control. However, relevant aspects of PCNA function are still not well understood, like the role of PCNA in the association with partner proteins, and how multiple protein interactions are orchestrated. Based on emerging evidence, I suggest that 1) PCNA interacting proteins may be reclassified in three major categories, namely, a) cell cycle control; b) DNA replication/repair; c) chromatin regulation/transcription. 2) PCNA is a negative regulator, rather than a processivity/recruitment factor, of chromatin-modifying enzymes. 3) At DNA replication sites, PCNA function may be envisaged with a model of "dynamic hand-off" of interacting partners that rapidly and transiently exchange in a mutually exclusive manner, while cyclin-dependent kinase (Cdk) 2 (CDK2) is stably bound to PCNA. The partner exchange might occur through a conformational change of the PCNA/protein/DNA complex allowing CDK2 to phosphorylate the partner protein, thereby enabling its hand-off from PCNA.

Cyclin-Dependent Kinase 2↗
PubMed1975

Procollagen assembly and secretion in embryonic chick bone.

Chick embryo skull bones incorporated radioactive proline and cystein into procollagen in short term organ culture. Pulse-chase experiments showed that individual precursor chains (pro-alpha1 and pro-alpha2) were formed first and that these were subsequently linked by disulfide bounds into trimers. Radioautography showed that labeled material was secreted 30 min after adding label to the cells, and electrophoretic analyses showed that after this time completed labeled collagen molecules appeared. Conversion from disulfide-linked procollagen to collagen proceeded in more than one step. An intermediate form consisting of shorter chains, which were still trimerically linked, was found.

Animals↗
PubMed2001

Solution structure of bacteriophage PRD1 vertex complex.

Bacteriophage PRD1 is a prototype of viruses with an internal membrane. The icosahedral capsid and major coat protein share structural similarity with the corresponding structures of adenovirus. The present study further explores similarities between these viruses, considering the 5-fold vertex assemblies. The vertex structure of bacteriophage PRD1 consists of proteins P2, P5, and P31. The vertex complex mediates host cell binding and controls double-stranded DNA delivery. Quaternary structures and interactions of purified spike proteins were studied by synchrotron radiation x-ray solution scattering. Low resolution models of the vertex proteins P5, P2, and P31 were reconstructed ab initio from the scattering data. Protein P5 is a long trimer that resembles the adenovirus spike protein pIV. The receptor-binding protein P2 is a 15.5-nm long, thin monomer and does not have an adenovirus counterpart. P31 forms a pentameric base with a maximum diameter of 8.5 nm, which is thinner than the adenovirus penton pIII. P5 further polymerize into a nonameric form ((P5(3))(3)). In the presence of P31, P5 associates into a P5(6):P31 complex. The constructed models of these assemblies provided support for a model of vertex assembly onto the virion. Although similar in overall architecture, clear differences between PRD1 and adenovirus spike assemblies have been revealed.

Bacteriophage PRD1↗
PubMed1985

Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity.

Laminin, a major structural glycoprotein of basement membranes, has been found to self-associate in vitro into large polymers. The formation of these complexes can be followed by the development of turbidity upon incubation in neutral phosphate buffer at 21-35 degrees C and is seen to be time-, concentration-, and temperature-dependent. The process is thermally reversible at 4 degrees C and the protein can be cycled between a dispersed and an aggregated state by alternating between 4 and 35 degrees C. Following incubation at 35 degrees C much of the monomeric laminin, which sediments at 11.4 S, is now seen to sediment at greater than 25 S. Both by turbidometric and sedimentation analysis, an apparent critical concentration for assembly of about 0.1 mg/ml (10(-7) M) is observed and is interpreted as evidence for a nucleation-propagation polymerization mechanism. The relative paucity of intermediates seen in a size-distribution analysis lends further support for this model. On platinum replicas obtained by rotary shadowing analysis, mostly free monomers are seen in the cold while after incubation at 35 degrees C, large multimeric aggregates with smaller amounts of oligomers are observed. The interaction between individual molecules appears to be specific because the dimers, trimers, and smaller oligomers are only associated at the terminal globular domains of the laminin molecules. In addition, removal of the globular domains of laminin with pepsin, which yields fragment P1, abolishes self-association. A divalent cation dependency for polymerization can be demonstrated and incubation in the presence of EDTA stops the polymerization at an oligomeric intermediate step. Hence overall laminin self-assembly can be divided into at least two steps: an initial temperature-dependent, divalent cation independent step followed by a divalent cation-dependent step.

Animals↗
PubMed2005

Structure and functional role of supercomplexes of IsiA and Photosystem I in cyanobacterial photosynthesis.

Cyanobacteria express large quantities of the iron stress-inducible protein IsiA under iron deficiency. IsiA can assemble into numerous types of single or double rings surrounding Photosystem I. These supercomplexes are functional in light-harvesting, empty IsiA rings are effective energy dissipaters. Electron microscopy studies of these supercomplexes show that Photosystem I trimers bind 18 IsiA copies in a single ring, whereas monomers may bind up to 35 copies in two rings. Work on mutants indicates that the PsaF/J and PsaL subunits facilitate the formation of closed rings around Photosystem I monomers but are not obligatory components in the formation of Photosystem I-IsiA supercomplexes.

Bacterial Proteins↗
PubMed1980

Aggregation of small oligonucleosomal chains into 300-A globular particles.

Chicken erythrocyte oligonucleosomes (trimers to about 20-mers) are able to interact with each other through the very lysine-rich histones (H1 and H5) and form heterogeneous globular particles with a mean diameter of about 300 A. These particles assemble spontaneously during micrococcal nuclease digestion of chromatin in the presence of 30 mM NaCl and contain approximately 25 nucleosomes. They are sensitive to ionic strength and unfold at lower salt concentrations but can be reconstituted by restoring the initial salt concentration. Even at 30 mM NaCl, the particles remain dynamic structures, being in equilibrium with their oligonucleosomal components as revealed by the fact that particle stability depends on the concentration of oligonucleosomes.

Animals↗