988 resultados para ionically bound proteins
Resumo:
We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed.
Resumo:
Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3′ ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative “bubble migration” mode of RDR could play a role in lesion bypass by the T4 replication machinery.
Resumo:
Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55/57 are required, whereas either Rad52 or Rad55/57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.
Resumo:
Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.
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Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}, which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-Å resolution the x-ray crystal structure of the complex of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg2+, and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅PSP and BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document}⋅CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.
Resumo:
The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.
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Constant pressure and temperature molecular dynamics techniques have been employed to investigate the changes in structure and volumes of two globular proteins, superoxide dismutase and lysozyme, under pressure. Compression (the relative changes in the proteins' volumes), computed with the Voronoi technique, is closely related with the so-called protein intrinsic compressibility, estimated by sound velocity measurements. In particular, compression computed with Voronoi volumes predicts, in agreement with experimental estimates, a negative bound water contribution to the apparent protein compression. While the use of van der Waals and molecular volumes underestimates the intrinsic compressibilities of proteins, Voronoi volumes produce results closer to experimental estimates. Remarkably, for two globular proteins of very different secondary structures, we compute identical (within statistical error) protein intrinsic compressions, as predicted by recent experimental studies. Changes in the protein interatomic distances under compression are also investigated. It is found that, on average, short distances compress less than longer ones. This nonuniform contraction underlines the peculiar nature of the structural changes due to pressure in contrast with temperature effects, which instead produce spatially uniform changes in proteins. The structural effects observed in the simulations at high pressure can explain protein compressibility measurements carried out by fluorimetric and hole burning techniques. Finally, the calculation of the proteins static structure factor shows significant shifts in the peaks at short wavenumber as pressure changes. These effects might provide an alternative way to obtain information concerning compressibilities of selected protein regions.
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Chemotaxis in bacteria is controlled by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein CheY. When phosphorylated, CheY binds to FliM, which is one of the proteins that constitute the "gear box" (or "switch") of the flagellar motor. Consequently, the motor shifts from the default direction of rotation, counterclockwise, to clockwise rotation. This biased rotation is terminated when CheY is dephosphorylated either spontaneously or, faster, by a specific phosphatase, CheZ. Logically, one might expect CheZ to act directly on FliM-bound CheY. However, here we provide direct biochemical evidence that, in contrast to this expectation, phosphorylated CheY (CheY approximately P), bound to FliM, is protected from dephosphorylation by CheZ. The complex between CheY approximately P and FliM was trapped by cross-linking with dimethylsuberimidate, and its susceptibility to CheZ was measured. CheY approximately P complexed with FliM, unlike free CheY approximately P, was not dephosphorylated by CheZ. However, it did undergo spontaneous dephosphorylation. Nonspecific cross-linked CheY dimers, measured as a control, were dephosphorylated by CheZ. No significant binding between CheZ and any of the switch proteins was detected. It is concluded that, in the termination mechanism of signal transduction in bacterial chemotaxis, CheZ acts only on free CheY approximately P. We suggest that CheZ affects switch-bound CheY approximately P by shifting the equilibrium between bound and free CheY approximately P.
Resumo:
The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.
Resumo:
All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.
Inhibition of phosphatidylinositol 3-kinase activity by association with 14-3-3 proteins in T cells.
Resumo:
Proteins of the 14-3-3 family can associate with, and/or modulate the activity of, several protooncogene and oncogene products and, thus, are implicated in regulation of signaling pathways. We report that 14-3-3 is associated with another important transducing enzyme, phosphatidylinositol 3-kinase (PI3-K). A recombinant 14-3-3 fusion protein bound several tyrosine-phosphorylated proteins from antigen receptor-stimulated T lymphocytes. PI3-K was identified by immunoblotting and enzymatic assays as one of the 14-3-3-binding proteins in resting or activated cells. Moreover, endogenous 14-3-3 and PI3-K were coimmunoprecipitated from intact T cells. Far-Western blots of gel-purified, immunoprecipitated PI3-K with a recombinant 14-3-3 fusion protein revealed direct binding of 14-3-3 to the catalytic subunit (p110) of PI3-K. Finally, anti-phosphotyrosine immunoprecipitates from activated, 14-3-3-overexpressing cells contained lower PI3-K enzymatic activity than similar immunoprecipitates from control cells. These findings suggest that association of 14-3-3 with PI3-K in hematopoietic (and possibly other) cells regulates the enzymatic activity of PI3-K during receptor-initiated signal transduction.
Resumo:
A monomorphic anti-HLA-G monoclonal antibody (mAb) was obtained by immunization of HLA-B27/human beta 2-microglobulin double-transgenic mice with transfected murine L cells expressing both HLA-G and human beta 2-microglobulin. This mAb, designated BFL.1, specifically recognizes, by flow cytometry analysis, the immunizing HLA-G-expressing cells, whereas it does not bind to parental untransfected or to HLA-B7- and HLA-A3-transfected L cells, suggesting that it distinguishes between classical HLA-A and -B and nonclassical HLA-G class I molecules. This was further assessed by the absence of BFL.1 reactivity with a number of human cell lines known to express classical HLA class I proteins. In addition, we showed that the BFL.1 mAb also labels HLA-G-naturally-expressing JEG-3 and HLA-G-transfected JAR human choriocarcinoma cell lines as well as a subpopulation of first-trimester placental cytotrophoblast cells. Further biochemical studies were performed by immunoprecipitation of biotinylated membrane lysates: BFL.1, like the monomorphic W6/32 mAb, immunoprecipitated a 39-kDa protein in HLA-G-expressing cell lines, a size corresponding to the predicted full-length HLA-G1 isoform. However, in contrast to W6/32, which immunoprecipitates both classical and nonclassical HLA class I heavy chains, BFL.1 mAb does not recognize the class Ia products. Such a mAb should be a useful tool for analysis of HLA-G protein expression in various normal and pathological human tissues and for determination of the function(s) of translated HLA-G products.
Resumo:
DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.
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The Saccharomyces cerevisiae gene ERD2 is responsible for the retrieval of lumenal resident proteins of the endoplasmic reticulum (ER) lost to the next secretory compartment. Previous studies have suggested that the retrieval of proteins by ERD2 is not essential. Here, we find that ERD2-mediated retrieval is not an essential process only because, on its failure, a second inducible system acts to maintain levels of ER proteins. The second system is controlled by the ER membrane-bound kinase encoded by IRE1. We conclude that IRE1 and ERD2 together maintain normal concentrations of resident proteins within the ER.
Resumo:
Two CO-isotope sensitive lines have been detected in the overtone region of the resonance Raman spectra of CO-bound hemeproteins. One line is assigned as the overtone of the Fe-CO stretching mode and is located in the 1000- to 1070-cm-1 region. The other line is found in the 1180- to 1210-cm-1 region and is assigned as a combination between a porphyrin mode, nu 7, and the Fe-CO stretching mode. The high intensities of these lines, which in the terminal oxidase class of proteins are of the same order as those of the fundamental stretching mode, indicate that the mechanism of enhancement for modes involving the Fe-CO moiety is different from that for the modes of the porphyrin macrocycle and call for reexamination of Raman theory of porphyrins as applied to axial ligands. The anharmonicity of the electronic potential function was evaluated, revealing that in the terminal oxidases the anharmonicity is greater than in the other heme proteins that were examined, suggesting a distinctive interaction of the bound CO with its distal environment in this family. Furthermore, the anharmonicity correlates with the frequency of the C-O stretching mode, demonstrating that both of these parameters are sensitive to the Fe-CO bond energy. The overtone and combination lines involving the bound CO promise to be additional probes of heme protein structural properties.