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Nucleic Acids Research Feb 1983E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S. Wickner, I.Berkower, M.Wright, and J. Hurwitz (1973) Proc. Natl. Acad....
E.coli dnaC protein was purified to near-homogeneity in using a dnaC complementation assay [S. Wickner, I.Berkower, M.Wright, and J. Hurwitz (1973) Proc. Natl. Acad. Sci. USA 70, 2369-2373]. Purification was achieved by taking advantage of the hydrophobic interaction of dnaC protein with aliphatic and aromatic matrixes and with Brij58 as stabilizing agent. A sedimentation coefficient for the dnaC protein of 2.6 S corresponding to a molecular weight of approximately 26,000 was estimated from glycerol gradient centrifugation. A polypeptide molecular weight of 28,000 was determined by densitometry on a denaturing gel. In the presence of ATP the dnaC protein forms a complex with dnaB protein [S. Wickner and J. Hurwitz (1975) Proc.Natl.Acad.Sci. USA 72, 921-925]. For the dnaB . dnaC complex a sedimentation coefficient of 14.5 S was measured by glycerol gradient centrifugation, indicating a molecular weight of about 400,000. The ratio of the dnaC and dnaB polypeptides in the complex is approximately 1, as determined on a denaturing gel. It is suggested that the complex consists of the dnaB protein hexamer and six dnaC polypeptides amounting to a calculated molecular weight of about 450,000.
Topics: Bacterial Proteins; Escherichia coli; Macromolecular Substances; Molecular Weight
PubMed: 6298736
DOI: 10.1093/nar/11.4.987 -
Cell Research Aug 2019ATM/Tel1 is an apical kinase that orchestrates the multifaceted DNA damage response. Mutations of ATM/Tel1 are associated with ataxia telangiectasia syndrome. Here, we...
ATM/Tel1 is an apical kinase that orchestrates the multifaceted DNA damage response. Mutations of ATM/Tel1 are associated with ataxia telangiectasia syndrome. Here, we report cryo-EM structures of symmetric dimer (4.1 Å) and asymmetric dimer (4.3 Å) of Saccharomyces cerevisiae Tel1. In the symmetric state, the side chains in Tel1 C-terminus (residues 1129-2787) are discernible and an atomic model is built. The substrate binding groove is completely embedded in the symmetric dimer by the intramolecular PRD and intermolecular LID domains. Point mutations in these domains sensitize the S. cerevisiae cells to DNA damage agents and hinder Tel1 activation due to reduced binding affinity for its activator Xrs2/Nbs1. In the asymmetric state, one monomer becomes more compact in two ways: the kinase N-lobe moves down and the Spiral of α-solenoid moves upwards, which resemble the conformational changes observed in active mTOR. The accessibility of the activation loop correlates with the synergistic conformational disorders in the TRD1-TRD2 linker, FATC and PRD domains, where critical post-translational modifications and activating mutations are coincidently condensed. This study reveals a tunable allosteric network in ATM/Tel1, which is important for substrate recognition, recruitment and efficient phosphorylation.
Topics: Allosteric Regulation; Ataxia Telangiectasia Mutated Proteins; Catalytic Domain; Cryoelectron Microscopy; DNA Breaks, Double-Stranded; DNA, Fungal; Dimerization; Intracellular Signaling Peptides and Proteins; Phosphorylation; Point Mutation; Protein Conformation, alpha-Helical; Protein Domains; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; TOR Serine-Threonine Kinases
PubMed: 31097817
DOI: 10.1038/s41422-019-0176-1 -
The Journal of Biological Chemistry Sep 1991An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where...
An enhancer-binding protein of the fibroin gene, fibroin factor 1 (FF1), has been purified to homogeneity from crude nuclear extracts of posterior silk gland cells where this gene is transcribed specifically. There is a multiplicity of FF1; the FF1 activity was eluted as at least three major fractions on column chromatographies. FF1 is able to form a stable complex with the enhancer DNA sequence in the presence of another proteinous factor named FF2, which lacks ability to bind DNA molecules by itself. One of FF1 forms, FF1a, was purified with a combination of classical purification techniques without using a sequence-specific affinity column, and identified as a protein with molecular mass 125 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To obtain homogeneous protein of FF1a, purification of more than 26,000-fold from the starting nuclear extract was necessary.
Topics: Animals; Base Sequence; Binding, Competitive; Bombyx; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; DNA; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Enhancer Elements, Genetic; Fibroins; Molecular Sequence Data
PubMed: 1885619
DOI: No ID Found -
Journal of Bacteriology Apr 2001The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four...
The glucan synthase complex of the human pathogenic mold Aspergillus fumigatus has been investigated. The genes encoding the putative catalytic subunit Fks1p and four Rho proteins of A. fumigatus were cloned and sequenced. Sequence analysis showed that AfFks1p was a transmembrane protein very similar to other Fksp proteins in yeasts and in Aspergillus nidulans. Heterologous expression of the conserved internal hydrophilic domain of AfFks1p was achieved in Escherichia coli. Anti-Fks1p antibodies labeled the apex of the germ tube, as did aniline blue fluorochrome, which was specific for beta(1-3) glucans, showing that AfFks1p colocalized with the newly synthesized beta(1-3) glucans. AfRHO1, the most homologous gene to RHO1 of Saccharomyces cerevisiae, was studied for the first time in a filamentous fungus. AfRho proteins have GTP binding and hydrolysis consensus sequences identical to those of yeast Rho proteins and have a slightly modified geranylation site in AfRho1p and AfRho3p. Purification of the glucan synthase complex by product entrapment led to the enrichment of four proteins: Fks1p, Rho1p, a 100-kDa protein homologous to a membrane H(+)-ATPase, and a 160-kDa protein which was labeled by an anti-beta(1-3) glucan antibody and was homologous to ABC bacterial beta(1-2) glucan transporters.
Topics: Amino Acid Sequence; Aspergillus fumigatus; Cloning, Molecular; Echinocandins; Fungal Proteins; Glucosyltransferases; Membrane Proteins; Molecular Sequence Data; Recombinant Proteins; Saccharomyces cerevisiae Proteins; rho GTP-Binding Proteins
PubMed: 11244067
DOI: 10.1128/JB.183.7.2273-2279.2001 -
Protein Science : a Publication of the... Sep 2005Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form...
Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.
Topics: Amino Acid Sequence; Blotting, Western; Congo Red; Endopeptidase K; Escherichia coli; Fluorescence; Microscopy, Electron; Molecular Sequence Data; Peptide Fragments; Peptide Hydrolases; Peptide Termination Factors; Prions; Saccharomyces cerevisiae Proteins; Temperature; Time Factors
PubMed: 16081647
DOI: 10.1110/ps.041234405 -
Cell Structure and Function Dec 1988Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these...
Several cell lines growing in protein- and lipid-free synthetic medium secreted cell-adhesive protein(s) into the medium. The conditioned medium (CM) of one of these cell lines, mouse L.P3, showed the highest cell attachment-promoting activity (CPA) among them. Cell-adhesive protein(s) in the CM of L.P3 cells (L.P3-CM) were separated into two types by sequential affinity column chromatography employing gelatin-Sepharose 4B and heparin-Sepharose 4B. One was a gelatin- and heparin-binding cell-adhesive protein (GCP), and was identified as a cellular form of mouse fibronectin. The other was a gelatin-non-binding and heparin-binding cell-adhesive protein (GNCP). The CPA of GNCP preparation was effective for the cell-attachment and spreading of both epithelial and fibroblastic cells. The CPA of GNCP preparation was not blocked by the antiserum and scarcely inhibited in the presence of the synthetic cell attachment-promoting peptide Gly-Arg-Gly-Asp-Ser-Pro, a competitive inhibitor of fibronectin. This suggests that the structure of the cell-attachment site of GNCP is different from that of fibronectin. The GNCP preparation showed little cross-reactivity with anti-mouse laminin antiserum in enzyme-linked immunosorbent assay (ELISA). These results demonstrate the possibility that GNCP in L.P3-CM is a novel cell-adhesive protein distinct from fibronectin or laminin. The secretion of the two types of cell-adhesive proteins by L.P3 cells is discussed.
Topics: Animals; Cell Adhesion; Cell Line; Chromatography, Affinity; Culture Media; Fibronectins; Lipids; Mice; Proteins
PubMed: 3242846
DOI: 10.1247/csf.13.481 -
Journal of Thrombosis and Haemostasis :... Feb 2006Plasma protein S (PS) is an essential anticoagulant that has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric... (Comparative Study)
Comparative Study
BACKGROUND AND OBJECTIVES
Plasma protein S (PS) is an essential anticoagulant that has activated protein C-independent, direct anticoagulant activity (PS-direct). It was reported that monomeric purified PS has poor PS-direct and that a subpopulation of multimeric purified PS has high PS-direct and high affinity for phospholipids. We independently examined the relative PS-direct and affinity for phospholipids of monomeric and multimeric PS and we obtained contrasting results.
METHODS AND RESULTS
Unpurified recombinant protein S (rPS) was monomeric and had PS-direct potency similar to that of both PS in plasma and multimeric affinity-purified PS, as measured in plasma assays for PS-direct and in thrombin-generation assays. Multimers of unpurified rPS were not induced by ethylenediaminetetraacetic acid (EDTA), pH 2.5, NaSCN, or barium adsorption/elution. Multimers were induced by chromatography in the presence of EDTA and thus may be concentration-dependent. In contrast to a different report, monomers, dimers, trimers, and higher-order PS forms were clearly separated in sedimentation velocity experiments and multimers were not dissociated by adding Ca(2+). Active plasma-derived and recombinant immunoaffinity-purified PS were fractionated into monomers and multimers. On a mass basis, monomers and multimers had similar specific PS-direct and ability to compete with prothrombinase components (factors Xa/Va) for limiting phospholipids. FXa ligand blotted to both monomers and multimers.
CONCLUSIONS
Plasma PS-direct is similar to that of affinity-purified PS and unpurified rPS. Under our conditions, monomeric and multimeric PS have similar PS-direct and ability to compete for phospholipids. Discordant earlier findings are likely due to loss of PS-direct during conventional purification procedures.
Topics: Anticoagulants; Binding, Competitive; Complement C4b-Binding Protein; Dimerization; Factor Xa; Histocompatibility Antigens; Humans; In Vitro Techniques; Ligands; Phospholipids; Protein S; Protein Structure, Quaternary; Recombinant Proteins; Ultracentrifugation
PubMed: 16420570
DOI: 10.1111/j.1538-7836.2006.01743.x -
Acta Crystallographica. Section F,... Jul 2012Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and...
Members of the Epsin protein family regulate the ubiquitin/clathrin-dependent trafficking of transmembrane proteins. The yeast Epsin-1 (ent1) gene was cloned and expressed in Escherichia coli. The protein product of a construct containing the ENTH-UIM modules was purified to homogeneity and subjected to crystallization screening using the sitting-drop vapour-diffusion method. Refined conditions containing polyethylene glycol 3350 and Tacsimate yielded thin rod-like crystals. X-ray analysis revealed that the crystallographic symmetry is primitive orthorhombic, space group P222, with unit-cell parameters a = 32.7, b = 35.5, c = 110.6 Å and a diffraction limit of 2.3 Å. Matthews coefficient calculations suggested that the crystal contained only the ENTH domain. This was corroborated by Coomassie Blue-stained SDS-PAGE analysis of dissolved crystals.
Topics: Cloning, Molecular; Crystallization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Vesicular Transport Proteins
PubMed: 22750874
DOI: 10.1107/S1744309112022488 -
Molecular BioSystems Apr 2011Four and a half LIM domains protein 1 (FHL1) is the most widely expressed member of the FHL family of proteins, consisting of four and a half highly conserved LIM...
INTRODUCTION
Four and a half LIM domains protein 1 (FHL1) is the most widely expressed member of the FHL family of proteins, consisting of four and a half highly conserved LIM domains. A multifunctional and integral role for FHL1 has been implicated in muscle development, structural maintenance, and signaling. To date, 27 FHL1 mutations have been identified that result in at least six different X-linked myopathies, with patients often presenting with cardiovascular complications. Since proteins assemble into dynamic complexes within the cell, FHL1 likely mediates its biological functions in conjunction with other proteins. Delineation of FHL1 interactions could provide insight into its regulatory functions.
METHODS
We performed tandem affinity purification from human embryonic kidney 293 (HEK-293) cells to purify FHL1 and interacting proteins. To identify the potential interactors of FHL1 we performed a total of 9 different purifications from HEK-293 cells which included 3 experimental replicates for each biological condition: FHL1, tag control (DPYSL3), and negative control (empty vector). Purified samples were analyzed by liquid chromatography mass spectrometry (LC-MS). Potential interactors were then verified by immunoprecipitation from mouse heart ventricles and interactions visualized in adult cardiomyocytes using 3D fluorescence microscopy.
RESULTS
We identified a total of 310 different proteins from all 9 purifications and by applying stringent filtering criteria we eliminated all proteins found in any of the controls and only allowed those that were detected in two or more bait purification. We identified 34 high confidence potential binding partners of FHL1. We then showed that FHL1 exists as part of a complex that binds with PDLIM1, GSN and ACTN1.
Topics: Actinin; Animals; Chromatography, Affinity; Computational Biology; Gelsolin; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Mass Spectrometry; Mice; Microfilament Proteins; Muscle Proteins; Protein Binding; Recombinant Fusion Proteins; Transcription Factors
PubMed: 21246116
DOI: 10.1039/c0mb00235f -
The Journal of Biological Chemistry Apr 1997The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively. Deletion of the VAM2 and VAM6 functions resulted in...
The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively. Deletion of the VAM2 and VAM6 functions resulted in accumulation of numerous vacuole-related structures of 200-400 nm in diameter that were much smaller than the normal vacuoles. Loss of functions of Vam2p and Vam6p resulted in inefficient processings of a set of vacuolar proteins, including proteinase A, proteinase B, and carboxypeptidase Y (CPY), and in severely defective maturation of another vacuolar protein, alkaline phosphatase. A part of newly synthesized CPY was missorted to the cell surface in the mutants. Epitope-tagged versions of Vam2p and Vam6p retained their functions, and they were found mostly in sedimentable fractions. The epitope-tagged Vam2p and Vam6p remained in the sedimentable fractions in the presence of Triton X-100, but they were extracted by urea or NaCl. Vam2p and Vam6p were cross-linked by the treatment of a chemical cross-linker. These observations indicated that Vam2p and Vam6p physically interact with each other and exist as components of a large protein complex. Vam6p fused with a green fluorescent protein were highly accumulated in a few specific regions of the vacuolar membranes. Large portions of Vam2p and Vam6p were fractionated into a vacuolar enriched fraction, indicating that they were localized mainly in the vacuolar membranes. These results showed that Vam2p and Vam6p execute their function in the vacuolar assembly as the components of a protein complex reside on the vacuolar membranes.
Topics: Amino Acid Sequence; Biological Transport; Carboxypeptidases; Carrier Proteins; Cathepsin A; Cell Compartmentation; Fungal Proteins; Genes, Fungal; Genomic Library; Intracellular Membranes; Membrane Proteins; Molecular Sequence Data; Mutation; Nuclear Proteins; Protein Precursors; Protein Processing, Post-Translational; RNA-Binding Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Analysis, DNA; Vacuoles; Vesicular Transport Proteins
PubMed: 9111041
DOI: 10.1074/jbc.272.17.11344