RESUMO
Reversible acetylation of core histone tails plays an important role in the regulation of eukaryotic transcription, in the formation of repressive chromatin complexes, and in the inactivation of whole chromosomes. The high-resolution X-ray structure of the nucleosome core particle, as well as earlier evidence, suggests that the histone tails are largely responsible for the assembly of nucleosomes into chromatin fibers and implies that the physiological effects of histone acetylation may be achieved by modulation of a dynamic inter-conversion between the fiber and a less condensed nucleofilament structure. In addition, the tails and adjacent regions serve as recognition sites for chromatin assembly and transcription remodeling machinery and the interactions that occur may also be responsive to histone acetylation.
Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Transcrição GênicaRESUMO
To facilitate the biochemical characterization of chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae, we have developed a system to assemble nucleosomal arrays on immobilized templates using recombinant yeast core histones. This system enabled us to analyze the interaction of Isw2 ATP-dependent chromatin remodeling complex with nucleosomal arrays. We found that Isw2 complex interacts efficiently with both naked DNA and nucleosomal arrays in an ATP-independent manner, suggesting that ATP is required at steps subsequent to this physical interaction. We identified the second subunit of Isw2 complex, encoded by open reading frame YGL 133w (herein named ITC1), and found that both subunits of the complex, Isw2p and Itc1p, are essential for efficient interaction with DNA and nucleosomal arrays. Both subunits are also required for nucleosome-stimulated ATPase activity and chromatin remodeling activity of the complex. Finally, we found that ITC1 is essential for function of Isw2 complex in vivo, since isw2 and itc1 deletion mutants exhibit virtually identical phenotypes. These results demonstrate the utility of our in vitro system in studying interactions between chromatin-associated proteins and nucleosomal arrays.
Assuntos
Adenosina Trifosfatases/genética , Cromatina/metabolismo , Histonas/genética , Nucleossomos/metabolismo , Fatores de Transcrição/genética , Leveduras/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Cromatina/genética , Cromatina/ultraestrutura , Estruturas Cromossômicas/genética , Estruturas Cromossômicas/metabolismo , Estruturas Cromossômicas/ultraestrutura , DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas Genéticas , Histonas/metabolismo , Dados de Sequência Molecular , Nucleossomos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Moldes Genéticos , Fatores de Transcrição/metabolismoRESUMO
The structural characterization of eukaryotic transcription factors that interact with DNA has advanced on two fronts in the past two years. New complexes of transcription factors bound to TATA-box DNA include the TFIIA-TBP-DNA complex as well as human and archaeal TBP-DNA and TFIIB-TBP-DNA complexes, respectively. In addition, recent studies of proximal/distal promoter complexes demonstrate that DNA-binding motifs may be modified in nature by adding secondary structure elements to diversify DNA-binding specificities and affinities.
Assuntos
Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Animais , Archaea/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Sequências Hélice-Alça-Hélice , Sequências Hélice-Volta-Hélice , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , TATA Box , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de ZincoRESUMO
The high resolution structure of the nucleosome core particle of chromatin reveals the form of DNA that is predominant in living cells and offers a wealth of information on DNA binding and bending by the histone octamer. Recent studies imply that chromatin is highly dynamic. This propensity for unfolding and refolding stems from the structural design of the nucleosome core. The histone-fold motif, central to nucleosome structure, is also found in other proteins involved in transcriptional regulation.
Assuntos
DNA/química , DNA/metabolismo , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Sequência de Aminoácidos , Animais , Archaea/química , Drosophila/química , Histonas/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Dobramento de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
An analytical formula has been derived for the calculation of the solvent accessible surface area of a protein molecule or equivalently the surface area exterior to an arbitrary number of overlapping spheres. The directional derivative of this function with respect to atomic co-ordinates is provided to facilitate minimization procedures used with molecular docking algorithms and energy calculations. An analytical formula for the calculation of the volume enclosed within the accessible surface, the excluded volume, is also derived. Although the area function is not specific to the structures of proteins, the derivation was motivated by the need for a computationally feasible simulation of the hydrophobic effect in proteins. A computer program using the equations for area has been tested and has had limited application to the docking of protein alpha-helices. Possible relationships of the solvent excluded volume to hydrophobic interaction free energy and transfer free energy of solute molecules are derived from the statistical mechanics of solution.
Assuntos
Proteínas , Solventes , Matemática , Modelos Químicos , Conformação Proteica , SoftwareRESUMO
The X-ray structure of the DNA binding domain of the yeast transcriptional activator protein GCN4 bound to a DNA fragment containing the sequence of the perfectly symmetrical ATF/CREB site has been solved to 3.0 A resolution. The architecture of this specific recognition complex supports the current model for bZIP proteins: a homodimer of parallel alpha-helices form an interhelix coiled-coil region via the leucine zipper, and the two N-terminal basic regions fit into the major groove of half sites on opposite sides of the DNA double helix. The structure shows that DNA flexibility plays the predominant role in the preservation of protein contacts with the symmetric ATF/CREB site (ATGACGTCAT) as compared to the pseudo-symmetric AP-1 target site (ATGACTCAT), overcoming the positional displacement of functional groups introduced by the additional G.C base-pair at the center of the ATF/CREB sequence.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas Fúngicas/química , Oligodesoxirribonucleotídeos/química , Proteínas Quinases/química , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/química , Fatores Ativadores da Transcrição , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Proteínas Sanguíneas , Cristalização , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fatores de Ligação G-Box , Genes Sintéticos , Zíper de Leucina , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/química , Proteínas de Plantas/química , Conformação Proteica , Proteínas Quinases/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Difração de Raios XRESUMO
Uniquely positioned nucleosomes were mapped in vitro on mouse mammary tumor 3' long terminal repeat (MMTV 3'LTR) DNA at base-pair resolution. Nucleosome A assembly was strongly favored over nucleosome B, and heating of each as a mononucleosome caused migration to the ends of the DNA fragment at a unique rate. Taken together with DNA sequence analysis, this suggests why MMTV 3'LTV nucleosome positions reported upstream of vector-derived sequences conflict and also how flanking genomic sequences could modulate the promoter in in vivo situations. Importantly, nucleosomes are shown to migrate for significant distances along DNA under physiologically relevant conditions, and the actual rates have been measured directly in solution. Exact positioning and shifting over greater than 60 bp has important consequences for transcription factor access to this MMTV promoter and for the role of nucleosomes in general.
Assuntos
Vírus do Tumor Mamário do Camundongo/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Sequências Repetitivas de Ácido Nucleico/genética , Adenina , Animais , Sequência de Bases , DNA Viral/química , Camundongos , Dados de Sequência Molecular , TiminaRESUMO
Members of the myocyte enhancer factor-2 (MEF2) family of transcription factors bind to and activate transcription through A+T-rich DNA sequences found primarily, but not exclusively, in the promoters of muscle-specific genes. Their importance has been established for myogenic development and in activation of the immediate-early gene, c-jun, and recently further functional roles in the immune system have emerged. The MEF2 factors belong to the MADS-box superfamily, sharing homology in a 58 amino acid domain that mediates DNA binding and dimerization. The structures of two MADS-box proteins, SRF and MCM1, bound to their cognate DNA have been previously reported and shown to share extensive similarity in their mode of DNA binding. We have solved the structure of MEF2A 2-78 bound to its DNA consensus sequence at 1.5 A resolution. It reveals how the absence of amino acids N-terminal to the MADS-box contributes to the DNA binding properties of MEF2 proteins and shows that the MEF domain C-terminal to the MADS-box adopts a conformation considerably different from the same region in SRF and MCM1.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Consenso/genética , Cristalografia por Raios X , DNA/química , DNA/genética , Dimerização , Ligação de Hidrogênio , Fatores de Transcrição MEF2 , Proteína 1 de Manutenção de Minicromossomo , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Regulação Miogênica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Elementos de Resposta/genética , Alinhamento de Sequência , Fator de Resposta Sérica , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Dimethyl sulfate, DNase I and micrococcal nuclease DNA cleavage were combined with the ligation-mediated polymerase chain reaction to obtain high resolution maps of the promoter regions for two cell-type-specific genes: the a-specific STE2 gene and the alpha-specific STE3 gene. We find that MCM1 binds in vivo in a-cells to a 16 bp P-box sequence located in the STE2 UAS. In alpha-cells, the footprint pattern is extended relative to a-cells, consistent with the additional binding of MAT alpha 2 to the sequences flanking each end of the P-box. A nucleosome was found adjacent to the P-box of the transcriptionally repressed a-specific STE2 UAS in alpha-cells, positioned so that the nucleosome overlaps the TATA-box. In contrast, such well-positioned nucleosomes were not found for the transcriptionally active STE2 UAS in a-cells, where instead the TATA box appears to be bound to the general transcription factor TFIID. These observations support the hypothesis that MAT alpha 2 repression of a-specific genes is mediated by nucleosomes, perhaps by exclusion of TFIID from the TATA-box.
Assuntos
Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Receptores de Peptídeos/genética , Receptores de Feromônios , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Sequência de Bases , Primers do DNA/química , Proteínas Fúngicas/genética , Genes Fúngicos , Dados de Sequência Molecular , Nucleossomos/ultraestrutura , RNA Mensageiro/genética , Receptores de Fator de Acasalamento , TATA BoxRESUMO
With the program FANTOM, we study the effect of a solvation energy term modelled by four atomic solvation parameter sets on energy refinement of proteins. Two parameter sets had previously been derived from measured free energies of transfer of hydrocarbons and amino acid side-chain analogues. Alternatively, the other two parameter sets correspond to the total or apolar accessible surface area of the protein. Twenty-five conformations of BPTI and the alpha-amylase inhibitor tendamistat were refined with respect to empirical energy terms (ECEPP/2) plus a solvation energy term modelled by one of the four atomic solvation parameter sets. These minimizations were compared to minimizations of the ECEPP/2 energy alone with regard to violations of upper distance limits obtained from NMR experiments as well as to root mean square deviations to NMR structures. We find that minimizations of the ECEPP/2 energy plus the total or apolar accessible surface area are superior to minimizations of the ECEPP/2 energy alone. In contrast, minimization of the ECEPP/2 energy plus a solvation energy term based on free energies of transfer perform poorly.
Assuntos
Conformação Proteica , Proteínas/química , Aprotinina/química , Simulação por Computador , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/química , Software , Solventes , Propriedades de SuperfícieRESUMO
The X-ray structure of the nucleosome core particle was determined at 7 A resolution using crystals containing mixed-sequence DNA and 21% to 27% of 1,6-hexanediol (partially dehydrated crystals). The alcohol was added to the crystals after growth to overcome the non-isomorphism of the crystals and improve the quality of their X-ray diffraction. Here, we report the structure of the nucleosome core particle from these crystals in the absence of the alcohol 1,6-hexanediol at 9 A resolution. The structure, under conditions of nearly full hydration, has been solved by multiple isomorphous replacement methods employing multiple heavy-atom compounds identical to those used for the partially dehydrated structure. The electron density of particles in the two crystal structures is well-correlated throughout the maps and structural elements of the DNA superhelix and histone proteins are generally similar, e.g. the DNA bends sharply at positions +/- 1 and +/- 4 double-helical turns from the DNA center. These results rule out the occurrence of gross structural changes in the 7 A structure due to addition of alcohol. The parts of the nucleosome core particle structure, which are dissimilar between the two forms, can be attributed to differences in molecular packing induced by the addition of 1,6-hexanediol. In contrast to the structure seen in the partially dehydrated crystals, the fully hydrated crystals show a particle in which the H2A-H2B dimers are symmetrically related by the dyad axis found in the H3-H4 tetramer region. However, in the fully hydrated crystals, the first and last double-helical turns of DNA superhelix are not related by dyad symmetry, and one of these segments has reduced contact with the adjacent H2A-H2B dimer.
Assuntos
Nucleossomos/química , Análise de Fourier , Água/química , Difração de Raios XRESUMO
General transcription factor IIF (TFIIF) is required for transcription by RNA polymerase II; it consists minimally of a heterodimer of RNA polymerase-associated proteins RAP30 and RAP74. According to solution and mutagenesis studies, the multiple domains of RAP30 and RAP74 bind PolII, TFIIB, TAF250 and DNA in interactions that are essential for transcription initiation and elongation. The X-ray structure of the RAP30/RAP74 interaction domains at 1.7 A resolution reveals a novel "triple barrel" dimerization fold and suggests with mutant data that interactions with the transcription apparatus are mediated not only by this tripartite beta-barrel, but also via flexible loops and alpha and beta-structures extending from it.
Assuntos
Dobramento de Proteína , Fatores de Transcrição TFII , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Endopeptidases/metabolismo , Humanos , Modelos Moleculares , Mutação , Maleabilidade , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas , Relação Estrutura-Atividade , Fatores de Transcrição/genéticaRESUMO
The X-ray structure of the GCN4-bZIP protein bound to DNA containing the ATF/CREB recognition sequence has been refined at 2.2 A. The water-mediated interactions between the basic domain and DNA are revealed, and combined with a more accurate description of the direct contacts, further clarify how binding specificity is achieved. Water molecules extend the interactions of both invariant basic domain residues, asparagine 235 and arginine 243, beyond their direct base contacts. The slight bending of the basic domain alpha-helix around the DNA facilitates the linking of arginine 241, 243 and 245 to main-chain carbonyl oxygen atoms via water molecules, apparently stabilizing interactions with the DNA.
Assuntos
Cristalografia por Raios X , Proteínas de Ligação a DNA/química , DNA/química , DNA/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina Básica , Sítios de Ligação , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fatores de Ligação G-Box , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Fatores de Transcrição/metabolismo , ÁguaRESUMO
Nucleosome core particles were reconstituted from a DNA restriction fragment and histone octamers, crystallized, and the crystals examined by X-ray diffraction. A DNA fragment was engineered by site-directed mutagenesis to obtain a 146 base-pair sequence that takes up a symmetrical arrangement in the core particle. The resulting DNA sequence was cloned in multiple copies into pUC9 and excised as monomer via EcoRV to produce it in milligram quantities. Nucleosome core particles incorporating the DNA were reconstituted by salt gradient dialysis and purified by anion-exchange high-pressure liquid chromatography. DNase I digestion was used to demonstrate that the termini of the restriction fragment are located 73 base-pairs from the molecular dyad axis of the particle. The diffraction limits of crystals of defined sequence core particles extend along the principal direction to a approximately equal to 4 A, b approximately equal to 5 A and c approximately equal to 3 A, giving about a twofold increase in the number of measurable X-ray reflections over previous crystals containing mixed sequence DNA. The methods developed here should be useful in the study of other large protein-DNA complexes.
Assuntos
DNA , Nucleossomos/ultraestrutura , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Cristalização , Dados de Sequência Molecular , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Difração de Raios XRESUMO
We describe our efforts to crystallize binary MCM1/DNA and ternary MATalpha2/MCM1/DNA complexes, including the unsuccessful attempts to crystallize MCM1/DNA complexes and the successful design of DNA crystal packing that resulted in high-resolution crystals of the MATalpha2/MCM1/DNA complex. We detail general procedures useful for preparing protein/DNA cocrystals, including improved methods for producing and purifying DNA-binding proteins and DNA fragments, for purifying protein/DNA complexes, and for controlling pH conditions during crystallization. We also describe the rational design of DNA for protein/DNA cocrystallization attempts, based on our analysis of how straight and bent DNA with single base-pair overhangs can pack end-to-end in a crystal.
Assuntos
DNA Fúngico/química , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Pareamento de Bases/genética , Sequência de Bases , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , DNA Recombinante/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/isolamento & purificação , Concentração de Íons de Hidrogênio , Proteína 1 de Manutenção de Minicromossomo , Modelos Moleculares , Peso Molecular , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/isolamento & purificação , Oligodesoxirribonucleotídeos/metabolismo , Regiões Operadoras Genéticas/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Plasmídeos/genética , Estrutura Terciária de Proteína , Receptores de Fator de Acasalamento , Receptores de Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Sefarose/análogos & derivados , Sefarose/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
In Xenopus somatic cells histone H1 effects the transcriptional repression of oocyte type 5 S RNA genes, without altering the transcription of the somatic type 5 S RNA genes. Using an unambiguous nucleosome mapping method we find substantial differences between the multiple in vitro nucleosome positions on the two types of genes. These nucleosome positions determine both transcription factor and H1 binding, allowing TFIIIA to bind more efficiently to nucleosomes containing the somatic 5 S RNA gene than to nucleosomes on the oocyte 5 S RNA gene. Significantly, in a binding competition between TFIIIA and H1, TFIIIA preferentially binds to the somatic nucleosome whereas H1 preferentially binds to the oocyte nucleosome, excluding TFIIIA binding. These results strongly suggest that nucleosome positioning plays a key role in the regulation of transcription of 5 S RNA genes and provide a molecular mechanism for the selective repression of the oocyte 5 S RNA genes by H1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Ribossômico 5S/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Feminino , Regulação da Expressão Gênica , Oócitos , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição TFIIIA , XenopusRESUMO
Crystals of mitochondrial creatine kinase isolated from chicken heart were grown by precipitation with polyethylene glycol 1000. The enzyme has been crystallized in the absence and presence of ATP in two different space groups. Crystals are tetragonal, with space group P42(1)2, a = b = 171 A, c = 150 A in the absence of ATP; and P422, a = b = 101 A, c = 114.4 A in the presence of ATP. We suggest that there is one octamer (346 kDa) per asymmetric unit without ATP and one dimer (86 kDa) per asymmetric unit with ATP. Using synchrotron radiation, the octameric form diffracts to at least 3 A resolution.
Assuntos
Creatina Quinase/ultraestrutura , Animais , Galinhas , Cristalografia , Mitocôndrias Musculares/enzimologia , Estrutura Molecular , Conformação Proteica , Difração de Raios XRESUMO
The X-ray structure of the nucleosome core particle was solved at 7 A resolution using the method of multiple isomorphous replacement based on two isomorphous derivatives, each containing a different multiple heavy-atom compound. The preparation of these heavy-atom compounds and their application to this macromolecular structure determination are described. The first of these reagents, TAMM (tetrakis(acetoxymercuri)methane), was solubilized by the addition of an excess of glycylglycine and, when added to crystals of the nucleosome core particle, produced a derivative with a single major site. Despite the large mass of 206,000 daltons per asymmetric unit, the position of the TAMM molecule was found in these crystals using the difference Patterson technique. This compound was sufficiently electron-dense to produce a unique solution, whereas the mono-mercurial, methylmercury nitrate had been inadequate. The second reagent, PIP (di-mu-iodobis(ethylenediamine)diplatinum(II) nitrate), is freely soluble in aqueous solution and, on addition to the crystals, labelled the histone proteins at several sites. The locations of the PIP groups were determined from difference Fourier and Patterson maps. The X-ray structure and solution characterization of this compound are reported. These multiple heavy-atom compounds appear to be generally applicable to X-ray structure determination, and are particularly useful in conjunction with crystals having asymmetric units of large volume but lacking non-crystallographic symmetry elements.
Assuntos
Compostos de Metilmercúrio , Nucleossomos/análise , Compostos Organoplatínicos , Cristalografia , Modelos MolecularesRESUMO
TlpA is an unusual thioredoxin-like protein present in the nitrogen-fixing soil bacterium Bradyrhizobium japonicum. A hydrophobic N-terminal transmembrane domain anchors it to the cytoplasmic membrane, whereby the hydrophilic thioredoxin domain becomes exposed to the periplasmic space. There, TlpA catalyses an essential reaction, probably a reduction, in the biogenesis of cytochrome aa(3). The soluble thioredoxin domain (TlpA(sol)), devoid of the membrane anchor, was purified and crystallized. Oxidized TlpA(sol) crystallized as a non-covalent dimer in the space group P2(1)2(1)2(1). The X-ray structure analysis was carried out by isomorphous replacement using a xenon derivative. This resulted in a high-resolution (1.6 A) three-dimensional structure that displayed all of the features of a classical thioredoxin fold. A number of peculiar structural details were uncovered: (i) Only one of the two active-site-cysteine sulphurs (Cys72, the one closer to the N terminus) is exposed on the surface, making it the likely nucleophile for the reduction of target proteins. (ii) TlpA(sol) possesses a unique structural disulphide bond, formed between Cys10 and Cys155, which connects an unprecedented N-terminal alpha helix with a beta sheet near the C terminus. (iii) An insertion of about 25 amino acid residues, not found in the thioredoxin prototype of Escherichia coli, contributes only marginally to the thioredoxin fold, but forms an extra, surface-exposed alpha helix. This region plus another surface-exposed stretch (-Ile-Gly-Arg-Ala-), which is absent even in the closest TlpA relatives, might be considered as specificity determinants for the recognition of target proteins in the periplasm. The TlpA(sol) structure paves the way towards unraveling important structure-function relationships by rational mutagenesis.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bradyrhizobium/química , Membrana Celular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Cisteína/metabolismo , Dissulfetos/metabolismo , Escherichia coli/química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Solubilidade , Eletricidade Estática , Relação Estrutura-Atividade , Xenônio/metabolismoRESUMO
The four core histone proteins, H2A, H2B, H3, and H4 of Xenopus laevis have been individually expressed in milligram quantities in Escherichia coli. The full-length proteins and the "trypsin-resistant" globular domains were purified under denaturing conditions and folded into histone octamers. Both intact and truncated recombinant octamers, as well as chicken erythrocyte octamer, were assembled into nucleosome core particles using a 146 bp defined-sequence DNA fragment from a 5 S RNA gene. The three types of core particles were characterized and compared by gel electrophoresis, DNase I cleavage, and tyrosine fluorescence emission during stepwise dissociation with increasing ionic strength. Nucleosome core particles containing native and mutant histones made in bacteria have facilitated its X-ray structure determination at 2.8 A resolution.