RESUMEN

Structures, stability and DNA-binding properties have been established for archaeal histones from mesophiles, thermophiles and hyperthermophiles. Most archaeal histones are simply histone folds that are stabilized by dimer formation. Archaeal histones and the histone folds of the eukaryotic nucleosome core histones share a common ancestry and bind and wrap DNA similarly using conserved residues. The histone-fold residues that stabilize dimer-dimer interactions within an archaeal histone core contribute to determining archaeal histone-DNA affinity.

Asunto(s)

Archaea/genética , Archaea/fisiología , ADN/química , Histonas/metabolismo , Secuencia de Aminoácidos , Archaea/química , Dicroismo Circular , Dimerización , Histonas/química , Calor , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/química , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido

RESUMEN

Lake Vostok, the largest subglacial lake in Antarctica, is separated from the surface by approximately 4 km of glacial ice. It has been isolated from direct surface input for at least 420 000 years, and the possibility of a novel environment and ecosystem therefore exists. Lake Vostok water has not been sampled, but an ice core has been recovered that extends into the ice accreted below glacial ice by freezing of Lake Vostok water. Here, we report the recovery of bacterial isolates belonging to the Brachybacteria, Methylobacterium, Paenibacillus and Sphingomonas lineages from a sample of melt water from this accretion ice that originated 3593 m below the surface. We have also amplified small-subunit ribosomal RNA-encoding DNA molecules (16S rDNAs) directly from this melt water that originated from alpha- and beta-proteobacteria, low- and high-G+C Gram-positive bacteria and a member of the Cytophaga/Flavobacterium/Bacteroides lineage.

Asunto(s)

Bacterias/aislamiento & purificación , Agua Dulce/microbiología , Hielo , Regiones Antárticas , Bacterias/clasificación , Bacterias/genética , Bacterias/ultraestructura , Fenómenos Fisiológicos Bacterianos , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , ADN Ribosómico/genética , ADN Ribosómico/aislamiento & purificación , Agua Dulce/química , Genes de ARNr , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Filogenia , Proteobacteria/clasificación , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , Proteobacteria/fisiología , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Microbiología del Agua

Asunto(s)

Archaea/genética , Cromosomas de Archaea/metabolismo , ADN de Archaea/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismo , Secuencia de Aminoácidos , Archaea/química , Archaea/metabolismo , Cromosomas de Archaea/genética , ADN de Archaea/genética , Evolución Molecular , Histonas/química , Histonas/genética , Datos de Secuencia Molecular

Asunto(s)

Archaea/química , ADN/metabolismo , Histonas/aislamiento & purificación , Nucleosomas/química , Proteínas Bacterianas/genética , Sitios de Unión , Heparina/química , Histonas/genética , Histonas/metabolismo , Nucleosomas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Here we describe the organization of the archaeal nucleosome, in which four archaeal histones are circumscribed by approximately 80 bp of DNA. Through a combination of sequence comparisons, 3D structural studies, site-directed mutagenesis and assays for DNA binding, we have assigned functions to most of the individual residues in the histone fold of the representative archaeal histone rHMfB. By SELEX selection, the sequences of DNA molecules that are most readily bound and wrapped by rHMfB into archaeal nucleosomes in vitro have been identified, and these define DNA structures that position archaeal nucleosome assembly.

Asunto(s)

Archaea/química , Archaea/genética , Histonas/metabolismo , Nucleosomas/genética , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Nucleosomas/metabolismo

RESUMEN

In the archaeon Methanobacterium thermoautotrophicum, MTH1669 encodes a protein with a sequence related to the N-terminal sequences of the alpha-subunits of eucaryal general transcription factor TFIIE. The recombinant MTH1669 gene product has been purified and shown to stimulate transcription in vitro from M. thermoautotrophicum promoters that were almost inactive or much less active in reaction mixtures that contained only M. thermoautotrophicum RNA polymerase, TATA-binding protein and transcription factor B. As all complete archaeal genome sequences contain an MTH1669 homolog, the protein encoded by this gene is apparently the first characterized example of a transcription activator, here designated TFE, that may be universally present in the Archaea.

Asunto(s)

Proteínas Arqueales/genética , Metano/metabolismo , Methanobacterium/genética , Factores de Transcripción TFII , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica Arqueal , Humanos , Hidrogenasas/genética , Hidrogenasas/metabolismo , Methanobacterium/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Moldes Genéticos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Dedos de Zinc/genética

RESUMEN

Archaeal histones and the eucaryal (eucaryotic) nucleosome core histones have almost identical histone folds. Here, we show that DNA molecules selectively incorporated by rHMfB (recombinant archaeal histone B from Methanothermus fervidus) into archaeal nucleosomes from a mixture of approximately 10(14) random sequence molecules contain sequence motifs shown previously to direct eucaryal nucleosome positioning. The dinucleotides GC, AA (=TT) and TA are repeated at approximately 10 bp intervals, with the GC harmonic displaced approximately 5 bp from the AA and TA harmonics [(GCN(3)AA or TA)(n)]. AT and CG were not strongly selected, indicating that TA not equalAT and GC not equalCG in terms of facilitating archaeal nucleosome assembly. The selected molecules have affinities for rHMfB ranging from approximately 9 to 18-fold higher than the level of affinity of the starting population, and direct the positioned assembly of archaeal nucleosomes. Fourier-transform analyses have revealed that AA dinucleotides are much enriched at approximately 10. 1 bp intervals, the helical repeat of DNA wrapped around a nucleosome, in the genomes of Eucarya and the histone-containing Euryarchaeota, but not in the genomes of Bacteria and Crenarchaeota, procaryotes that do not have histones. Facilitating histone packaging of genomic DNA has apparently therefore imposed constraints on genome sequence evolution, and since archaeal histones have no structure in addition to the histone fold, these constraints must result predominantly from histone fold-DNA contacts. Based on the three-domain universal phylogeny, histones and histone-dependent genome sequence evolution most likely evolved after the bacterial-archaeal divergence but before the archaeal-eucaryal divergence, and were subsequently lost in the Crenarchaeota. However, with lateral gene transfer, the first histone fold could alternatively have evolved after the archaeal-eucaryal divergence, early in either the euryarchaeal or eucaryal lineages.

Asunto(s)

Archaea , ADN/genética , Evolución Molecular , Genoma Bacteriano , Histonas/metabolismo , Nucleosomas/genética , Archaea/química , Archaea/citología , Archaea/genética , Secuencia de Bases , Clonación Molecular , ADN/química , ADN/metabolismo , Huella de ADN , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Repeticiones de Dinucleótido/genética , Células Eucariotas , Análisis de Fourier , Regulación de la Expresión Génica Arqueal , Genoma Arqueal , Histonas/química , Nucleasa Microcócica/metabolismo , Datos de Secuencia Molecular , Nucleosomas/química , Nucleosomas/metabolismo , Filogenia

RESUMEN

The hyperthermophilic archaeon Methanothermus fervidus contains two small basic proteins, HMfA (68 amino acid residues) and HMfB (69 residues) that share a common ancestry with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. HMfA and HMfB have sequences that differ at 11 locations, they have different structural stabilities, and the complexes that they form with DNA have different electrophoretic mobilities. Here, crystal structures are documented for recombinant (r) HMfA at a resolution of 1.55 A refined to a crystallographic R-value of 19.8 % (tetragonal form) and at 1.48 A refined to a R-value of 18.8 % (orthorhombic form), and for rHMfB at 1.9 A refined to a R-value of 18.0 %. The rHMfA and rHMfB monomers have structures that are just histone folds in which a long central alpha-helix (alpha2; 29 residues) is separated from shorter N-terminal (alpha1; 11 residues) and C-terminal (alpha3; 10 residues) alpha-helices by two loops (L1 and L2; both 6 residues). Within L1 and L2, three adjacent residues are in extended (beta) conformation. rHMfA and rHMfB assemble into homodimers, with the alpha2 helices anti-parallel aligned and crossing at an angle of close to 35 degrees, and with hydrogen bonds formed between the extended, parallel regions of L1 and L2 resulting in short beta-ladders. Dimerization creates a novel N-terminal structure that contains four proline residues, two from each monomer. As prolines are present at these positions in all archaeal histone sequences, this proline-tetrad structure is likely to be a common feature of all archaeal histone dimers. Almost all residues that participate in monomer-monomer interactions are conserved in HMfA and HMfB, consistent with the ability of these monomers to form both homodimers and (HMfA+HMfB) heterodimers. Differences in side-chain interactions that result from non-conservative residue differences in HMfA and HMfB are identified, and the structure of a (rHMfA)(2)-DNA complex is presented based on the structures documented here and modeled by homology to histone-DNA interactions in the eukaryal nucleosome.

Asunto(s)

Histonas/química , Methanobacteriales/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN de Archaea/química , ADN de Archaea/genética , ADN de Archaea/metabolismo , Dimerización , Electroforesis en Gel de Agar , Histonas/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Prolina/química , Prolina/metabolismo , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Electricidad Estática , Xenopus laevis , Zinc/metabolismo

RESUMEN

A decade after the discovery of histones in Archaea, there is now also a biochemical description of the archaeal nucleosome. A tetrameric core of archaeal histones is encircled by approximately 80 bp of DNA, and nuclease digestions indicate that adjacent archaeal nucleosomes exist in vivo compacting archaeal genomic DNA. Most Eukarya employ a similar structure to organize their chromosomal DNA, the eukaryal nucleosome, with a histone octamer and 146 bp of DNA. Here we compare the properties of both nucleosomes in terms of DNA packaging and the accessibility of the packaged DNA for transcription.

Asunto(s)

Archaea/genética , Histonas , Nucleosomas , Secuencia de Aminoácidos , Células Eucariotas , Regulación de la Expresión Génica Arqueal , Datos de Secuencia Molecular , Células Procariotas

RESUMEN

Site-specific mutagenesis of the hmfB gene cloned from the archaeon Methanothermus fervidus, followed by expression in Escherichia coli, has been used to generate approximately 90 recombinant (r) variants of the archaeal histone HMfB. The abilities of these variants to form stable archaeal nucleosome-containing complexes with linear pBR322 DNA, and with an 89 bp restriction fragment of this DNA have been determined. Variants that failed to form such complexes, based on negative gel-shift assays, had substitutions at the N terminus or within the alpha1, L1 and L2 regions of the rHMfB histone fold, at sites predicted to be homologous to eucaryal histone fold residues that contact the DNA in the eucaryal nucleosome. Variants that failed to give gel shifts were further assayed for their abilities to facilitate ligase-catalyzed circularization of a linear 88 bp DNA molecule, and to reduce the ellipticity of a DNA solution at 275 nm (theta(275)). Consistent with cooperative but independent sites of DNA binding, a combination of three residue substitutions, one each in alpha1, L1 and L2, was required to generate a rHMfB variant with no detectable DNA binding based on gel shift, circularization and theta(275) reduction assays.

Asunto(s)

Proteínas Arqueales/metabolismo , ADN/metabolismo , Histonas/metabolismo , Methanobacteriales/genética , Mutación/genética , Sitio Alostérico , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Dicroismo Circular , ADN/química , ADN/genética , ADN Ligasas/metabolismo , ADN Circular/química , ADN Circular/genética , ADN Circular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Electroforesis , Genes Arqueales/genética , Variación Genética/genética , Histonas/química , Histonas/genética , Histonas/aislamiento & purificación , Methanobacteriales/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia

RESUMEN

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three alpha-helices separated by two beta-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647delta) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)4 tetramer, were apparently less stable than those formed by (rMJ1647delta)4. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647delta in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones.

Asunto(s)

Proteínas Arqueales/genética , Histonas/química , Methanococcus/genética , Sistemas de Lectura Abierta , Secuencia de Aminoácidos , Proteínas Arqueales/química , Dicroismo Circular , Clonación Molecular , Proteínas de Unión al ADN/química , Dimerización , Escherichia coli , Methanococcus/química , Datos de Secuencia Molecular , Rotación Óptica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Alineación de Secuencia , Temperatura , Termodinámica

RESUMEN

Amino acid residues responsible for the large difference in thermostability between HMfB and HFoB, archaeal histones from the hyperthermophile Methanothermus fervidus and the mesophile Methanobacterium formicicum, respectively, have been identified by site-specific mutagenesis. The thermal denaturation of approximately 70 archaeal histone variants has been monitored by circular dichroism, and the data generated were fit to a two-state unfolding model (dimer-->two random coil monomers) to obtain a standard-state (1M) melting temperature for each variant dimer. The results of single-, double-, and triple-residue substitutions reveal that the much higher stability of rHMfB dimers, relative to rHFoB dimers, is conferred predominantly by improved intermolecular hydrophobic interactions near the center of the histone dimer core and by additional favorable ion pairs on the dimer surface.

Asunto(s)

Histonas/genética , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Dimerización , Histonas/química , Calor , Methanobacterium , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína

RESUMEN

RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum DeltaH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

Asunto(s)

Proteínas Arqueales , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación de la Expresión Génica Arqueal , Methanobacterium/genética , Factor de Transcripción TFIIB , Transcripción Genética , Secuencia de Aminoácidos , Centrifugación por Gradiente de Densidad , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Hidrógeno/metabolismo , Metano/metabolismo , Methanobacterium/enzimología , Methanobacterium/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pruebas de Precipitina , Proteína de Unión a TATA-Box , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

Asunto(s)

Archaea/clasificación , Archaea/genética , Archaea/patogenicidad , ARN de Archaea/genética

RESUMEN

DNA in Methanothermus fervidus, a hyperthermophilic archaeon, is constrained into archaeal nucleosomes in vivo by the archaeal histones HMfA and HMfB. Here, we document the translational and rotational positioning of archaeal nucleosome assembly in vitro by a sequence from the 7S RNA encoding region of the M. fervidus genome. The minor groove of the DNA at the center of the DNA sequence, protected from micrococcal nuclease digestion by incorporation into a positioned archaeal nucleosome, faces away from the archaeal histone core.

Asunto(s)

Archaea/genética , ADN de Archaea , Methanobacteriales/genética , Nucleosomas/genética , Secuencia de Bases , Datos de Secuencia Molecular , Biosíntesis de Proteínas

RESUMEN

DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)6 and (CTG)8 repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)4-, (CTG)5-, or (CTG)3AA(CTG)3-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.

Asunto(s)

Archaea/genética , ADN de Archaea/genética , Nucleosomas/fisiología , Repeticiones de Trinucleótidos , Archaea/metabolismo , Secuencia de Bases , Clonación Molecular , ADN de Archaea/química , Escherichia coli , Histonas/metabolismo , Datos de Secuencia Molecular , Mapeo Restrictivo , Moldes Genéticos

RESUMEN

Archaeal histones, homologs of the eucaryal nucleosome core histones, have been identified in the Euryarchaeota. They assemble as tetramers with dsDNA to form archaeal nucleosomes that resemble the central structure of the eucaryal nucleosome formed by the histone (H3-H4)2 tetramer. Eucaryal and archaeal nucleosomes assemble preferentially on DNA molecules that best accommodate the severe bends found within these structures, and here we discuss the relationships between archaeal and eucaryal nucleosomes, repeating DNA sequences, and nucleosome positioning.

Asunto(s)

Archaea/genética , Genoma Arqueal , Nucleosomas/química , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Archaea/ultraestructura , Núcleo Celular/genética , ADN de Archaea/análisis , ADN de Archaea/química , ADN de Archaea/genética , Repeticiones de Dinucleótido , Electroforesis en Gel de Poliacrilamida , Células Eucariotas/ultraestructura , Histonas/genética , Datos de Secuencia Molecular , ARN de Archaea/análisis , ARN Ribosómico/análisis , Alineación de Secuencia , Secuencias Repetidas en Tándem , Repeticiones de Trinucleótidos

RESUMEN

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.

Asunto(s)

ADN de Archaea/metabolismo , ADN Circular/metabolismo , Histonas/análisis , Methanobacteriales , Nucleosomas/química , ADN Superhelicoidal , Conformación de Ácido Nucleico , Unión Proteica

RESUMEN

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3 + H4)2 tetramer.

Asunto(s)

Archaea/química , Archaea/genética , Histonas/química , Histonas/genética , Secuencia de Aminoácidos , Animales , ADN de Archaea/química , Dimerización , Células Eucariotas , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Nucleosomas/química , Conformación Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Transcripción Genética

RESUMEN

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is > 90% reversible, with concentration-dependent apparent Tm values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyA1 dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing < 200 mM, < 400 mM, and < 1.5 M KCl, respectively. rHMfA, rHMfB, and rHFoB also experience significant cold denaturation in low salt concentrations and at low pH. The midpoint of thermal unfolding of a 1 M protein solution (T degree value) and the temperature dependency of the free energy of unfolding have been established for each histone, and both parameters correlate with the growth temperature of the originating archaeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtained by altering the curvature of the stability curve. Rather, the stability curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher Tmax values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyA1 are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degree values for rHFoB, rHMfA, rHMfB, and rHPyA1 are 75, 104, 113, and 114 degrees C, respectively, at pH 5 in 0.2 M KCl. Structural features within the conserved histone fold that might confer these stability differences are discussed.

Asunto(s)

Histonas/química , Methanobacterium/química , Pyrococcus/química , Termodinámica , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Dimerización , Histonas/genética , Concentración de Iones de Hidrógeno , Methanobacterium/genética , Modelos Químicos , Datos de Secuencia Molecular , Pliegue de Proteína , Pyrococcus/genética , Proteínas Recombinantes/química , Sales (Química) , Temperatura