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PubMed2004

Whole-genome shotgun assembly and comparison of human genome assemblies.

We report a whole-genome shotgun assembly (called WGSA) of the human genome generated at Celera in 2001. The Celera-generated shotgun data set consisted of 27 million sequencing reads organized in pairs by virtue of end-sequencing 2-kbp, 10-kbp, and 50-kbp inserts from shotgun clone libraries. The quality-trimmed reads covered the genome 5.3 times, and the inserts from which pairs of reads were obtained covered the genome 39 times. With the nearly complete human DNA sequence [National Center for Biotechnology Information (NCBI) Build 34] now available, it is possible to directly assess the quality, accuracy, and completeness of WGSA and of the first reconstructions of the human genome reported in two landmark papers in February 2001 [Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A., Holt, R. A., et al. (2001) Science 291, 1304-1351; International Human Genome Sequencing Consortium (2001) Nature 409, 860-921]. The analysis of WGSA shows 97% order and orientation agreement with NCBI Build 34, where most of the 3% of sequence out of order is due to scaffold placement problems as opposed to assembly errors within the scaffolds themselves. In addition, WGSA fills some of the remaining gaps in NCBI Build 34. The early genome sequences all covered about the same amount of the genome, but they did so in different ways. The Celera results provide more order and orientation, and the consortium sequence provides better coverage of exact and nearly exact repeats.

Computational Biology↗
PubMed2006

Structure and assembly of P-pili: a protruding hinge region used for assembly of a bacterial adhesion filament.

High-resolution structures of macromolecular complexes offer unparalleled insight into the workings of biological systems and hence the interplay of these systems in health and disease. We have adopted a multifaceted approach to understanding the pathogenically important structure of P-pili, the class I adhesion pili from pyelonephritic Escherichia coli. Our approach combines electron cryomicroscopy, site-directed mutagenesis, homology modeling, and energy calculations, resulting in a high-resolution model of PapA, the major structural element of these pili. Fitting of the modeled PapA subunit into the electron cryomicroscopy data provides a detailed view of these pilins within the supramolecular architecture of the pilus filament. A structural hinge in the N-terminal region of the subunit is located at the site of a newly resolved electron density that protrudes from the P-pilus surface. The structural flexibility provided by this hinge is necessary for assembly of P-pili, illustrating one solution to construction of large macromolecular complexes from small repeating units. These data support our hypothesis that domain-swapped pilin subunits transit the outer cell membrane vertically and rotate about the hinge for final positioning into the pilus filament. Our data confirm and supply a structural basis for much previous genetic, biochemical, and structural data. This model of the P-pilus filament provides an insight into the mechanism of assembly of a macromolecular complex essential for initiation of kidney infection by these bacteria.

Bacterial Adhesion↗
PubMed1976

Protein cleavage during virus assembly: a novel specificity of assembly dependent cleavage in bacteriophage T4.

Cleavage of precursor proteins occurs during assembly of numerous viruses. Seven bacteriophage T4 head-related proteins areknown to be cleaved during morphogenesis. Sequences surrounding the cleavage sites in T4 head precursors P23 and IPIII are reported here. We previously determined the sequences of precursor and processed forms of IPII and IPI. Cleavage occurs at a glutamyl-alanyl bond in each protein. By comparison of sequences around five cleaved and four uncleaved Glu-Ala bonds in head precursors, it appears that cleavage is limited to the Thr or Ala, and X2 to hydrophilic residues. The results suggest the viral-induced assembly protease recognizes and cleaves an extended primary structure in the structurally dissimilar precursors.

Amino Acid Sequence↗
PubMed1996

The ordered assembly of the phiX174-type primosome. II. Preservation of primosome composition from assembly through replication.

Gel filtration chromatography was used to isolate both preprimosomal and primosomal complexes formed on single-stranded DNA-binding protein-coated phiX174 DNA by the combination of PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG. The presence and relative amounts of primosomal proteins in these complexes were determined by Western blotting. Protein-DNA complexes isolated (i) after assembly in the presence of 10 microM ATP, (ii) after preprimosome movement in the presence of 1 mM ATP, (iii) after priming in the presence of the four ribonucleoside triphosphates, or (iv) after complementary strand DNA replication in the presence of the DNA polymerase III holoenzyme all had the same protein composition; preprimosomes contained PriA, PriB, PriC, DnaT, and DnaB, whereas primosomes included DnaG. The stable association of DnaG with the protein-DNA complex could be attributed partially to its ability to remain bound to the primers synthesized. In the absence of PriC, the efficiencies of priming and replication were reduced by one-third and one-half, respectively, even though PriC was not required for the formation of stable protein-DNA complexes on a 304-nucleotide-long single strand of DNA containing a primosome assembly site (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15642-15648). We hypothesize that maintenance of the primosome on the replicated DNA may provide a mechanism to allow primosomes to participate in the resolution of recombination intermediates and intermediates formed during double strand break repair by permitting the re-establishment of a replication fork.

Bacterial Proteins↗
PubMed1997

Collagen II containing a Cys substitution for arg-alpha1-519. Homotrimeric monomers containing the mutation do not assemble into fibrils but alter the self-assembly of the normal protein.

A recombinant system was used to prepare human type II procollagen containing the substitution of Cys for Arg at alpha1-519 found in three unrelated families with early onset generalized osteoarthritis together with features of a mild chondrodysplasia probably best classified as spondyloepiphyseal dysplasia. In contrast to mutated procollagens containing Cys substitutions for obligatory Gly residues, the Cys substitution at alpha1-519 did not generate any intramolecular disulfide bonds. The results were consistent with computer modeling experiments that demonstrated that the alpha carbon distances were shorter with Cys substitutions for obligatory Gly residues than with Cys substitutions in the Y position residues in repeating -Gly-X-Y- sequences of the collagen triple helix. The mutated collagen did not assemble into fibrils under conditions in which the normal monomers polymerized. However, the presence of the mutated monomer in mixtures with normal collagen II increased the lag time for fibril assembly and altered the morphology of the fibrils formed.

Amino Acid Sequence↗
PubMed2005

Trapping of an assembly intermediate of photosynthetic LH1 antenna beyond B820 subunit. Significance for the assembly of photosynthetic LH1 antenna.

Most photosynthetic LH1 antennae undergo dissociation into B820 subunits, suggesting their universal character as structural modules. However, dissociation into subunits seems to occur reversibly only in the absence of carotenoids and the subunits were never found to bind carotenoids. The interactions of carotenoids with B820 have been studied in a newly developed reconstitution assay of the LH1 antenna from Rhodospirillum rubrum (Fiedor, L., Akahane, J., and Koyama, Y. (2004) Biochemistry 43, 16487-16496). These model studies show that B820 subunits strongly interact with carotenoids and spontaneously form stable LH1-like complexes with substoichiometric carotenoid content. This is the first experimental evidence that B820 may occur as a short-lived intermediate in the assembly of LH1 in vivo. The resulting complex of B820 subunits with carotenoid, termed iB873, is homogeneous, according to ion exchange chromatography and reproducible pigment composition. The iB873-bound carotenoid is as efficient in energy transfer to bacteriochlorophyll as the one in native antenna. To our knowledge, iB873 is the first complex binding functional carotenoid, with the spectral and biochemical properties intermediate between that of B820 and the fully assembled LH1.

Acetone↗
PubMed1973

The synthesis, assembly, and secretion of gamma globulin by mouse myeloma cells. VI. Assembly of IgM proteins.

The study of the synthesis, assembly, and secretion of IgM by seven murine myeloma tumors has revealed that free mu chain can be detected intracellularly after release from the ribosome. It combines with light chains to form microL. The major intracellular protein in six of the seven tumors was the 8S subunit. One tumor contained considerable amounts of 19S material intracellularly. Those tumors that did not contain 19S IgM intracellulariy appeared to assemble the subunits outside the cell.

Animals↗
PubMed2004

ThurGood: evaluating assembly-to-assembly mapping.

The alignment and mapping of large genomic sequences is the focus of much recent research. However, relatively little has been done so far about testing and validating alignment methods. We introduce criteria and new tools we have developed for alignment evaluation. These tools have already proved useful in the evaluation and ranking of several methods for assembly-to-assembly mapping, which were recently used to map multiple versions of the human genome to each other (Istrail et aL, 2004).

Algorithms↗
PubMed2006

Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro.

An amorphous silica mineralization technique was used to produce inorganic/protein composites to elucidate the structure and mechanism of formation of amelogenin assemblies, which may play an important role in regulating enamel structure during the initial stages of amelogenesis. Full-length recombinant amelogenins from mouse (rM179) and pig (rP172) were investigated along with key degradation products (rM166 and native P148) lacking the hydrophilic C terminus found in parent molecules. The resulting products were examined using transmission electron microscopy and/or small-angle X-ray scattering. Using protein concentrations of 0.1-3 mg ml-1, large monodisperse spheres of remarkably similar mean diameters were observed using rM179 (124+/-4 nm) and rP172 (126+/-7 nm). These spheres also exhibited 'internal structure', comprising nearly spherical monodisperse particles of approximately 20 nm in diameter. In the presence of rM166, P148, and bovine serum albumin (control), large unstructured and randomly shaped particles (250-1000 nm) were observed. Without added protein, large dense spherical particles of silica (mean approximately 500 nm) lacking internal structure were produced. These findings demonstrate that full-length amelogenins have the ability to form higher-order structures, whereas amelogenins that lack the hydrophilic C terminus do not. The results also suggest that full-length amelogenin can guide the formation of organized mineralized structures through co-operative interactions between assembling protein and forming mineral.

Amelogenesis↗
PubMed1989

Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface.

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium-dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro-GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.

Antibodies, Monoclonal↗
PubMedDate not supplied

A switch in disulfide linkage during minicollagen assembly in hydra nematocysts or how to assemble a 150-bar-resistant structure.

Hydra minicollagen, the shortest collagen known, is an important component of the nematocyst wall, which has a very high tensile strength. It has an unusual structure, with small and closely related Cys-rich domains at both ends of its chains. Three chains are trimerized by a central collagenous domain. Polyhydroxyproline helices connect the Cys-rich domains with the collagenous domain. The minicollagen precursor contains three internal disulfide bridges in each Cys-rich domain and no disulfide bridges between chains of the same trimeric molecule or between different molecules. Biochemical and structural evidence as well as confocal immunofluorescence microscopy points to disulfide-mediated assembly during maturation of nematocysts.

Animals↗
PubMed1994

Assembly and molecular organization of self-assembled lipid bilayers on solid substrates monitored by surface plasmon resonance spectroscopy.

The structural properties of lipid films, made from a squalene/butanol solution containing varying amounts (0-15 mg/ml) of egg phosphatidylcholine and deposited on a thin metallic silver layer, were investigated using surface plasmon resonance (SPR) spectroscopy. Optical parameters (thickness, refractive index and extinction coefficient) of such supported self-assembled lipid membranes were obtained from a theoretical analysis of the experimental SPR curves. The mass of the lipid membrane and the area and volume occupied by one lipid molecule were also calculated. The results were consistent with the formation of durable and homogeneous lipid bilayers on the solid substrate, and indicated similarities in structural properties between the present lipid bilayers and freely suspended and Langmuir-Blodgett bilayer membranes. Such bilayers represent a simple model for biological membranes, as well as providing a means of immobilizing proteins for various practical applications, including receptor-based sensors and molecular devices. The results confirm the value of the SPR technique for investigating the properties of thin biomolecular dielectric films deposited on a metal surface.

Lipid Bilayers↗
PubMed1988

Primate cytomegalovirus assembly: evidence that DNA packaging occurs subsequent to B capsid assembly.

Results presented here show that when cytomegalovirus (strain Colburn)-infected cells are treated with the DNA synthesis inhibitor hydroxyurea or phosphonoformate, one type of intranuclear capsid accumulates. These particles appeared to contain symmetrically organized internal material, and had a protein composition and sedimentation rate characteristic of B capsids. Radiolabeling experiments provided evidence that a population of B capsids lacking DNA is present during the course of a normal infection. These capsids sedimented slightly slower than the peak of viral DNA in the same region of the gradient, and had a ratio of DNA/protein that was estimated to be sevenfold lower than that of the faster sedimenting C capsids. DNA in both the B and C capsid regions of such gradients was found to be relatively resistant to digestion with DNase. The possibility is considered that herpesvirus B capsids lacking DNA may be counterparts of unexpanded proheads in the bacteriophage assembly pathway.

Capsid↗
PubMed1981

Membrane assembly from purified components. II. Assembly of M13 procoat into liposomes reconstituted with purified leader peptidase.

The major coat protein of coliphage M13 is an integral protein of the E. coli plasma membrane prior to its assembly into new virus particles. It is generated from its precursor, procoat, by a membrane-bound leader peptidase. We now describe the reconstitution of a highly purified preparation of this enzyme into vesicles of E. coli phospholipids. These vesicles bind procoat made in vitro and procoat isolated from in vitro synthesis. Both the crude and the purified substrates were converted post-translationally to coat protein. A significant proportion of the coat protein becomes inserted into the vesicle bilayer, with the N terminus facing the vesicle interior and the C terminus exposed to the external medium. These results strongly suggest that highly purified leader peptidase from E. coli and phospholipids are the only components necessary to mediate the binding, processing and insertion of this integral membrane protein.

Coliphages↗
PubMed1987

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures.

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.

Adenosine Diphosphate↗
PubMed2002

Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts.

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.

Actin-Related Protein 2↗
PubMed2004

Structure and electrochemistry of 4,4'-dithiodipyridine self-assembled monolayers in comparison with 4-mercaptopyridine self-assembled monolayers on Au(111).

4,4'-Dithiodipyridine (PySSPy) monolayers on Au(111) were investigated by cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and in situ scanning tunneling microscopy (STM). The studies were performed in solutions of different anions and pHs (0.1 M H2SO4, 0.1 M HClO4, 0.1 and 0.01 M Na2SO4, 0.1 and 0.01 M NaOH). The cyclic current-potential curves in H2SO4 show current peaks at about 0.4 V, which are absent for all other electrolytes at this potential. The XPS data suggest that PySSPy adsorbs via the S endgroup on the gold surface and the S-S bond breaks during adsorption. From the chemical shift of the N(ls) peak, it is concluded that in acidic media the self-assembled monolayer (SAM) is fully protonated, whereas in basic solution it is not. The pKa is estimated to be 5.3. STM studies reveal the existence of highly ordered superstructures for the SAM. In Na2SO4 and H2SO4, a (7 x mean square root of 3) structure is proposed. However, whereas in Na2SO4 solutions the superstructure does not change with potential, in 0.1 M H2SO4 the superstructure is observed only negative of the current peak at +0.4 V. At more positive potentials, the film becomes disordered. The results are compared to those for 4-mercaptopyridine (PyS) SAMs. XPS experiments and current-potential curves indicate that both molecules adsorb in the same manner on Au(111), that is, even in the case of PySSPy the adspecies is PyS. The STM results, however, call for a more subtle interpretation. While in Na2SO4 solutions the observed superstructures are the same for both SAMs, markedly different structures are found for PySSPy and PyS SAMs in 0.1 M H2SO4.

Journal Article↗
PubMed2001

Discrete versus infinite molecular self-assembly: control in crystalline hydrogen-bonded assemblies based on resorcinol.

Cocrystallization of 1,2-bis(4-pyridyl)ethane (4,4'-bipyeth) with resorcinol (res), 4-chlororesorcinol (4-Cl-res), and 4,6-dichlororesorcinol (4,6-di-Cl-res) yields molecular solids (4,4'-bipyeth).(res) 1a, 2(4,4'-bipyeth).2(4-Cl-res) 1b, and 2(4,4'-bipyeth).2(4,6-di-Cl-res) 1c with components held together by O-H...N hydrogen bonds. In 1a the components form an infinite 1D polar array, whereas in 1b and 1c the components form 0D four-component complexes. Formation of the discrete assemblies is attributed to peripheral steric effects, which block the solid-state polymerization. [reaction: see text]

Journal Article↗