Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Chem Commun (Camb) ; 52(69): 10537-9, 2016 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-27491621

RESUMO

The chaperonin thermosome (THS) is a protein cage that lacks binding sites for metal ions and inorganic nanoparticles. However, when poly(amidoamine) (PAMAM) is encapsulated into THS, gold nanoparticles (AuNP) can be prepared in the THS. The polymer binds HAuCl4. Subsequent reduction yields nanoparticles with narrow size distribution in the protein-polymer conjugate.


Assuntos
Cloretos/química , Dendrímeros/química , Compostos de Ouro/química , Ouro/química , Nanopartículas Metálicas/química , Poliaminas/química , Termossomos/química , Tamanho da Partícula , Thermoplasma
2.
Angew Chem Int Ed Engl ; 53(5): 1443-7, 2014 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-24459061

RESUMO

The group II chaperonin thermosome (THS) from the archaea Thermoplasma acidophilum is reported as nanoreactor for atom-transfer radical polymerization (ATRP). A copper catalyst was entrapped into the THS to confine the polymerization into this protein cage. THS possesses pores that are wide enough to release polymers into solution. The nanoreactor favorably influenced the polymerization of N-isopropyl acrylamide and poly(ethylene glycol)methylether acrylate. Narrowly dispersed polymers with polydispersity indices (PDIs) down to 1.06 were obtained in the protein nanoreactor, while control reactions with a globular protein-catalyst conjugate only yielded polymers with PDIs above 1.84.


Assuntos
Chaperoninas/metabolismo , Radicais Livres/química , Nanotecnologia , Termossomos/metabolismo , Acrilamidas/química , Acrilamidas/metabolismo , Catálise , Chaperoninas/química , Cobre/química , Ligantes , Poliaminas/química , Poliaminas/metabolismo , Polimerização , Thermoplasma/metabolismo , Termossomos/química
3.
Protein Cell ; 4(6): 432-44, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23709365

RESUMO

Group II chaperonins, which assemble as double-ring complexes, assist in the refolding of nascent peptides or denatured proteins in an ATP-dependent manner. The molecular mechanism of group II chaperonin assembly and thermal stability is yet to be elucidated. Here, we selected the group II chaperonins (cpn-α and cpn-ß), also called thermosomes, from Acidianus tengchongensis and investigated their assembly and thermal stability. We found that the binding of ATP or its analogs contributed to the successful assembly of thermosomes and enhanced their thermal stabilities. Cpn-ß is more thermally stable than cpn-α, while the thermal stability of the hetero thermosome cpn-αß is intermediate. Cryo-electron microscopy reconstructions of cpn-α and cpn-ß revealed the interwoven densities of their non-conserved flexible N/C-termini around the equatorial planes. The deletion or swapping of their termini and pH-dependent thermal stability assays revealed the key role of the termini electrostatic interactions in the assembly and thermal stability of the thermosomes.


Assuntos
Termossomos/metabolismo , Acidianus/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Nucleotídeos/metabolismo , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Temperatura , Termossomos/química , Termossomos/genética
4.
Biophys J ; 103(6): 1285-95, 2012 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-22995501

RESUMO

Chaperonins are molecular machines that use ATP-driven cycles to assist misfolded substrate proteins to reach the native state. During the functional cycle, these machines adopt distinct nucleotide-dependent conformational states, which reflect large-scale allosteric changes in individual subunits. Distinct allosteric kinetics has been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL, undergo concerted subunit motions within each ring, whereas archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. We study these distinct mechanisms through a comparative normal mode analysis of monomer and double-ring structures of the archaeal chaperonin thermosome and GroEL. We find that thermosome monomers of each type exhibit common low-frequency behavior of normal modes. The observed distinct higher-frequency modes are attributed to functional specialization of these subunit types. The thermosome double-ring structure has larger contribution from higher-frequency modes, as it is found in the GroEL case. We find that long-range intersubunit correlation of amino-acid pairs is weaker in the thermosome ring than in GroEL. Overall, our results indicate that distinct allosteric behavior of the two chaperonin classes originates from different wiring of individual subunits as well as of the intersubunit communications.


Assuntos
Proteínas Arqueais/química , Modelos Moleculares , Termossomos/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Proteínas Arqueais/metabolismo , Mathanococcus , Movimento , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Termossomos/metabolismo
5.
J Struct Biol ; 180(1): 249-53, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22584152

RESUMO

Chemical biotinylation of protein complexes followed by binding to two-dimensional (monolayer) crystals of streptavidin is shown to be an effective way to prepare cryo-EM specimens from samples at low protein concentration. Three different multiprotein complexes are used to demonstrate the generality of this method. In addition, native thermosomes, purified from Sulfolobus solfataricus P2, are used to demonstrate that a uniform distribution of Euler angles is produced, even though this particle is known to adopt a preferred orientation when other methods of cryo-EM specimen preparation are used.


Assuntos
Biotina/química , Microscopia Crioeletrônica/métodos , Estreptavidina/química , Adsorção , Animais , Apoferritinas/química , Apoferritinas/ultraestrutura , Proteínas de Bactérias/química , Biotinilação , Cristalização , Desulfovibrio vulgaris , Cavalos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/ultraestrutura , Ligação Proteica , Estrutura Quaternária de Proteína , Sulfolobus solfataricus , Termossomos/química , Termossomos/ultraestrutura
6.
Chimia (Aarau) ; 65(4): 245-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21678771

RESUMO

Damage self-reporting materials are able to indicate the presence of microscopic damaged regions by easy to detect signals, such as fluorescence. Therefore, these smart materials can reduce the risk of catastrophic failure of load-bearing components, e.g., in aerospace and construction applications. We highlight here our proof-of-concept paper and we present some additional data, which shows that proteins can be used as mechanophores in solid polymeric materials. Macroscopic mechanical forces were transferred from the polymer to the embedded proteins. The biomolecules act as molecular strain sensor, giving the material the desired self-reporting property. Poly(ethylene glycol) and poly(acrylamide) (PAAm) networks were doped with small amounts of thermsosome (THS), a protein cage from the family of chaperonins, that encapsulated a pair of fluorescent proteins. THS acts as a scaffold which brings the two fluorescent proteins into distance suitable for fluorescence resonance energy transfer (FRET). Moreover, THS can be distorted by mechanic forces so that the distance between the fluorescent proteins changes, leading to a change in FRET efficiency. Using the brittle PAAm as a model system, we were able to visualize microcracks in the polymers by FRET microscopy and by fluorescence lifetime imaging. THS also stabilizes the encapsulated guest proteins against thermal denaturation, increasing their half-live at 70 degrees C by a factor of 2.3.


Assuntos
Resinas Acrílicas/química , Proteínas de Bactérias/química , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Polietilenoglicóis/química , Termossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência
7.
Biochem Soc Trans ; 39(1): 94-8, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21265753

RESUMO

It is now well understood that, although proteins fold spontaneously (in a thermodynamic sense), many nevertheless require the assistance of helpers called molecular chaperones to reach their correct and active folded state in living cells. This is because the pathways of protein folding are full of traps for the unwary: the forces that drive proteins into their folded states can also drive them into insoluble aggregates, and, particularly when cells are stressed, this can lead, without prevention or correction, to cell death. The chaperonins are a family of molecular chaperones, practically ubiquitous in all living organisms, which possess a remarkable structure and mechanism of action. They act as nanoboxes in which proteins can fold, isolated from their environment and from other partners with which they might, with potentially deleterious consequences, interact. The opening and closing of these boxes is timed by the binding and hydrolysis of ATP. The chaperonins which are found in bacteria are extremely well characterized, and, although those found in archaea (also known as thermosomes) and eukaryotes have received less attention, our understanding of these proteins is constantly improving. This short review will summarize what we know about chaperonin function in the cell from studies on the archaeal chaperonins, and show how recent work is improving our understanding of this essential class of molecular chaperones.


Assuntos
Archaea/metabolismo , Chaperoninas/metabolismo , Termossomos/metabolismo , Chaperoninas/química , Chaperoninas/genética , Microscopia Crioeletrônica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformação Proteica , Dobramento de Proteína , Termossomos/química , Termossomos/genética , Termossomos/ultraestrutura
8.
J Struct Biol ; 173(1): 77-85, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20719249

RESUMO

Classification and averaging of sub-tomograms can improve the fidelity and resolution of structures obtained by electron tomography. Here we present a three-dimensional (3D) maximum likelihood algorithm--MLTOMO--which is characterized by integrating 3D alignment and classification into a single, unified processing step. The novelty of our approach lies in the way we calculate the probability of observing an individual sub-tomogram for a given reference structure. We assume that the reference structure is affected by a 'compound wedge', resulting from the summation of many individual missing wedges in distinct orientations. The distance metric underlying our probability calculations effectively down-weights Fourier components that are observed less frequently. Simulations demonstrate that MLTOMO clearly outperforms the 'constrained correlation' approach and has advantages over existing approaches in cases where the sub-tomograms adopt preferred orientations. Application of our approach to cryo-electron tomographic data of ice-embedded thermosomes revealed distinct conformations that are in good agreement with results obtained by previous single particle studies.


Assuntos
Algoritmos , Interpretação Estatística de Dados , Tomografia com Microscopia Eletrônica/métodos , Tomografia com Microscopia Eletrônica/estatística & dados numéricos , Modelos Moleculares , Termossomos/química , Tomografia com Microscopia Eletrônica/classificação , Funções Verossimilhança
9.
Structure ; 18(10): 1270-9, 2010 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-20947016

RESUMO

Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpnß) in the open state from Acidianus tengchongensis. There is a ∼30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnß were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.


Assuntos
Proteínas Arqueais/química , Conformação Proteica , Termossomos/química , Acidianus/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Clonagem Molecular , Microscopia Crioeletrônica , Cristalização , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Termossomos/genética , Termossomos/metabolismo , Difração de Raios X
10.
Biochem Biophys Res Commun ; 393(2): 228-34, 2010 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-20117082

RESUMO

Recombinant thermosomes from the Acidianus tengchongensis strain S5(T) were purified to homogeneity and assembled in vitro into homo-oligomers (rATcpnalpha or rATcpnbeta) and hetero-oligomers (rATcpnalphabeta). The symmetries of these complexes were determined by electron microscopy and image analysis. The rATcpnalpha homo-oligomer was shown to possess 8-fold symmetry while both rATcpnbeta and rATcpnalphabeta oligomers adopted 9-fold symmetry. rATcpnalphabeta oligomers were shown to contain the alpha and beta subunits in a 1:2 ratio. All of the complexes prevented the irreversible inactivation of yeast alcohol dehydrogenase at 55 degrees C and completely prevented the formation of aggregates during thermal inactivation of citrate synthase at 45 degrees C. All rATcpn complexes showed trace ATP hydrolysis activity. Furthermore, rATcpnbeta sequestered fully chemically denatured substrates (GFP and thermophilic malic dehydrogenase) in vitro without refolding them in an ATP-dependent manner. This property is similar to previously reported properties of chaperonins from Sulfolobus tokodaii and Sulfolobus acidocaldarius. These features are consistent with the slow growth rates of these species of archaea in their native environment.


Assuntos
Acidianus/metabolismo , Peptídeos/metabolismo , Termossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Hidrólise , Microscopia Eletrônica , Peptídeos/química , Peptídeos/genética , Filogenia , Dobramento de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfolobus/metabolismo , Sulfolobus acidocaldarius/metabolismo , Termossomos/química , Termossomos/genética
11.
Pac Symp Biocomput ; : 252-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19908377

RESUMO

TRiC is an important group II chaperonin that facilitates the folding of many eukaryotic proteins. The TRiC complex consists of two stacked rings, each comprised of eight paralogous subunits with a mutual sequence identity of 30-35%. Each subunit has unique functional roles that are manifested by corresponding sequence conservation. It is generally assumed that the subunit order within each ring is fixed, but this order is still uncertain. Here we address the problem of the intra-ring subunit order by combining two sources of information: evolutionary conservation and a structural hypothesis. Specifically, we identify residues in the TRiC subunits that are likely to be part of the intra-unit interface, based on homology modeling to the solved thermosome structure. Within this set of residues, we search for a subset that shows an evolutionary conservation pattern that is indicative of the subunit order key. This pattern shows considerable conservation across species, but large variation across the eight subunits. By this approach we were able to locate two parts of the interface where complementary interactions seem to favor certain pairing of subunits. This knowledge leads to restrictions on the 5,040 (=7!) possible subunits arrangements in the ring, and limits them to just 72. Although our findings give only partial understanding of the inter-subunit interactions that determine their order, we conclude that they are comprised of complementary charged, polar and hydrophobic interactions that occur in both the equatorial and middle domains of each subunit.


Assuntos
Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Animais , Biologia Computacional , Sequência Conservada , Evolução Molecular , Humanos , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Termossomos/química , Termossomos/genética
12.
Mol Microbiol ; 74(5): 1152-68, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19843217

RESUMO

Chaperonins are macromolecular machines that assist in protein folding. The archaeon Methanosarcina mazei has acquired numerous bacterial genes by horizontal gene transfer. As a result, both the bacterial group I chaperonin, GroEL, and the archaeal group II chaperonin, thermosome, coexist. A proteome-wide analysis of chaperonin interactors was performed to determine the differential substrate specificity of GroEL and thermosome. At least 13% of soluble M. mazei proteins interact with chaperonins, with the two systems having partially overlapping substrate sets. Remarkably, chaperonin selectivity is independent of phylogenetic origin and is determined by distinct structural and biochemical features of proteins. GroEL prefers well-conserved proteins with complex alpha/beta domains. In contrast, thermosome substrates comprise a group of faster-evolving proteins and contain a much wider range of different domain folds, including small all-alpha and all-beta modules, and a greater number of large multidomain proteins. Thus, the group II chaperonins may have facilitated the evolution of the highly complex proteomes characteristic of eukaryotic cells.


Assuntos
Proteínas Arqueais/metabolismo , Chaperoninas do Grupo I/metabolismo , Chaperoninas do Grupo II/metabolismo , Methanosarcina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/análise , Proteínas Arqueais/química , Proteínas Arqueais/genética , Chaperonina 60/genética , Chaperonina 60/metabolismo , Células Eucarióticas/metabolismo , Chaperoninas do Grupo I/química , Chaperoninas do Grupo I/genética , Chaperoninas do Grupo II/química , Chaperoninas do Grupo II/genética , Methanosarcina/genética , Modelos Moleculares , Filogenia , Ligação Proteica/genética , Dobramento de Proteína , Proteoma/análise , Especificidade por Substrato , Termossomos/química , Termossomos/genética , Termossomos/metabolismo
13.
Protein Eng Des Sel ; 22(10): 607-13, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19622546

RESUMO

Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 beta-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Cinética , Modelos Moleculares , Penicilina Amidase/química , Penicilina Amidase/genética , Penicilina Amidase/metabolismo , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Termossomos/química , Termossomos/genética , Termossomos/metabolismo , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo
15.
Biotechnol Bioeng ; 102(2): 417-24, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18846552

RESUMO

We have previously shown that a single-subunit thermosome from Methanocaldococcus jannaschii (rTHS) can stabilize enzymes in semi-aqueous media (Bergeron et al., 2008b). In the present study, rTHS was used to stabilize penicillin amidase (PGA) in methanol-water mixtures. Including methanol in the reaction medium for amoxicillin synthesis can suppress unwanted hydrolysis reactions but inactivate PGA. Inactivation and reactivation pathways proposed for PGA illustrate the predictability of enzyme stabilization by rTHS in co-solvents. Calcium was necessary for reversible dissociation of the two PGA subunits in methanol-water and the presence of calcium resulted in an enhancement of chaperone-assisted stabilization. rTHS also acted as a stabilizer in the enzymatic synthesis of the beta-lactam antibiotic amoxicillin. rTHS stabilized PGA, increasing its half-life in 35% methanol by fivefold at 37 degrees C. Stabilization by rTHS was enhanced but did not require the presence of ATP. Including rTHS in fed-batch reactions performed in methanol-water resulted in nearly 4 times more amoxicillin than when the reaction was run without rTHS, and over threefold higher selectivity towards amoxicillin synthesis compared to aqueous conditions without rTHS. The thermosome and other thermophilic chaperones may thus be generally useful for stabilizing enzymes in their soluble form and expanding the range of conditions suitable for biocatalysis.


Assuntos
Amoxicilina/metabolismo , Antibacterianos/biossíntese , Proteínas Arqueais/metabolismo , Chaperoninas/metabolismo , Chaperonas Moleculares/metabolismo , Penicilina Amidase/metabolismo , Trifosfato de Adenosina/metabolismo , Estabilidade Enzimática , Temperatura Alta , Mathanococcus/metabolismo , Penicilina Amidase/antagonistas & inibidores , Termossomos
16.
Arch Biochem Biophys ; 481(1): 45-51, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18976628

RESUMO

The functionality of regions within the equatorial domain of Group II chaperonins is poorly understood. Previously we showed that a 70 amino acid sequence within this domain on the single-subunit recombinant thermosome from Methanocaldococcus jannaschii (rTHS) contains residues directly responsible for refolding protein substrates [L.M. Bergeron, C. Lee, D.S. Clark, Identification of a critical chaperoning region on an archaeal recombinant thermosome, Biochem. Biophys. Res. Commun. 369 (2008) 707-711]. In the present study, 6-aminopenicillanic acid (6-APA) was found to bind to rTHS and inhibit it from refolding proteins. Fluorescence anisotropy was used to measure a 6-APA/rTHS dissociation constant of 17.1 microM and verify that the binding site is within the first 70 amino-terminal rTHS residues. Docking simulations point to a specific loop region at residues 53-57 on rTHS as the most likely binding region. This loop region is located within the oligomeric association sites of the wild-type thermosome. These results implicate a specific equatorial region of Group II chaperonins in the refolding of proteins, and suggest its importance in conformational changes that accompany chaperone function.


Assuntos
Proteínas Arqueais/química , Chaperoninas/química , Methanococcaceae/metabolismo , Chaperonas Moleculares/química , Polarização de Fluorescência , Modelos Moleculares , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Termossomos
17.
Biochem Biophys Res Commun ; 369(2): 707-11, 2008 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-18313393

RESUMO

Chaperone function in water-miscible organic co-solvents is useful for biocatalytic applications requiring enzyme stability in semi-aqueous media and for understanding chaperone behavior in hydrophobic environments. Previously, we have shown that a recombinant single subunit thermosome (rTHS) from Methanocaldococcus jannaschii functions in multiple co-solvents to hydrolyze ATP, prevent protein aggregation, and refold enzymes following solvent denaturation. For the present study, a truncated analog to the thermosome in which 70 N-terminal amino acids are removed is used to identify important regions within the thermosome for its chaperoning functions in organic co-solvents. Data presented herein indicate that the N-terminal region of rTHS is essential for the chaperone to restore the native state of the enzyme citrate synthase, but it is not a critical region for either binding of unfolded proteins or ATP hydrolysis. This is the first demonstration that direct refolding by a Group II chaperonin requires the N-terminal region of the protein.


Assuntos
Trifosfato de Adenosina/química , Proteínas Arqueais/química , Proteínas Arqueais/ultraestrutura , Chaperoninas/química , Chaperoninas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Chaperonas Moleculares/química , Proteínas Arqueais/genética , Chaperoninas/genética , Simulação por Computador , Chaperonas Moleculares/genética , Dobramento de Proteína , Proteínas Recombinantes/química , Termossomos
18.
FEMS Microbiol Lett ; 266(1): 103-9, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17092293

RESUMO

The gene encoding for a putative thermosome from the hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE0907) from A. pernix K1 showed some homology with other group II chaperonins from archaea. The recombinant ApcpnA protein has a molecular mass of 60 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exhibited ATPase activity with an optimum temperature and pH of 90 degrees C and 5.0, respectively. The ATPase activity was found to be dependent on manganese and potassium ions, but not magnesium ion. The K(m) for ATP at pH 5.0 and 90 degrees C was 10.04 (+/- 1.31) microM, and k(cat) was determined to be 2.21 (+/- 0.11) min(-1) for the ApcpnA monomer. The recombinant ApcpnA prevents thermal aggregation of bovine rhodanese and enhances the thermal stability of alcohol dehydrogenase in vitro, indicating that the protein is suitable as a molecular chaperonin in the high-temperature environment.


Assuntos
Aeropyrum/metabolismo , Proteínas Arqueais/fisiologia , Chaperoninas/fisiologia , Subunidades Proteicas/fisiologia , Temperatura , Aeropyrum/genética , Álcool Desidrogenase/metabolismo , Animais , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Bovinos , Chaperoninas/química , Chaperoninas/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/metabolismo , Genes Arqueais , Cinética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Termossomos , Tiossulfato Sulfurtransferase/metabolismo
19.
J Mol Biol ; 362(4): 835-43, 2006 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-16942780

RESUMO

Using a combination of intrinsic fluorescence to report ATP-induced rearrangements, quenched-flow to measure ATP hydrolysis "on-enzyme" and optical methods to probe the kinetics of product release, we have begun to dissect the process of energy transduction in the thermosome, a type II chaperonin from Thermoplasma acidophilum. Stoichiometric measurements of ATP binding reveal the tight association of eight nucleotide molecules per hexa-decamer, implying the filling of only one ring owing to strong negative cooperativity. After binding, we show that these eight ATP molecules are hydrolysed over the next 50 s, after which hydrolysis slows down markedly during the establishment of the steady state in the ATPase reaction, demonstrating that the kinetic system is off-rate limited. Looking in more detail, this rapid first-turnover can be dissected into two phases; the first occurring with a half-time of 0.8 s, the second with a half-time of 14 s, possibly reflecting the differential behaviour of the four alpha and four beta subunits in a single thermosome ring. To investigate the post-hydrolytic events, we used two heat-stable enzyme-linked optical assays to measure the rate of evolution of ADP and of phosphate from the thermosome active site. Neither product showed a rapid dissociation phase prior to the establishment of the steady state, showing that both are released slowly at a rate that limits the cycle. These data highlight the importance of the highly populated thermosome/ADP/Pi complex in the molecular mechanism.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Arqueais/metabolismo , Chaperoninas/metabolismo , Thermoplasma/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Fluorescência , Hidrólise , Cinética , Fosfatos/metabolismo , Ligação Proteica , Termossomos
20.
Proc Natl Acad Sci U S A ; 102(20): 7303-8, 2005 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-15883368

RESUMO

Based primarily on 16S rRNA sequence comparisons, life has been broadly divided into the three domains of Bacteria, Archaea, and Eukarya. Archaea is further classified into Crenarchaea and Euryarchaea. Archaea generally thrive in extreme environments as assessed by temperature, pH, and salinity. For many prokaryotic organisms, ribosomal proteins (RP), transcription/translation factors, and chaperone genes tend to be highly expressed. A gene is predicted highly expressed (PHX) if its codon usage is rather similar to the average codon usage of at least one of the RP, transcription/translation factors, and chaperone gene classes and deviates strongly from the average gene of the genome. The thermosome (Ths) chaperonin family represents the most salient PHX genes among Archaea. The chaperones Trigger factor and HSP70 have overlapping functions in the folding process, but both of these proteins are lacking in most archaea where they may be substituted by the chaperone prefoldin. Other distinctive PHX proteins of Archaea, absent from Bacteria, include the proliferating cell nuclear antigen PCNA, a replication auxiliary factor responsible for tethering the catalytic unit of DNA polymerase to DNA during high-speed replication, and the acidic RP P0, which helps to initiate mRNA translation at the ribosome. Other PHX genes feature Cell division control protein 48 (Cdc48), whereas the bacterial septation proteins FtsZ and minD are lacking in Crenarchaea. RadA is a major DNA repair and recombination protein of Archaea. Archaeal genomes feature a strong Shine-Dalgarno ribosome-binding motif more pronounced in Euryarchaea compared with Crenarchaea.


Assuntos
Archaea/genética , Proteínas Arqueais/metabolismo , Chaperoninas/metabolismo , Expressão Gênica , Genoma Arqueal , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Arqueais/genética , Chaperoninas/genética , Códon/genética , Proteínas Ribossômicas/genética , Termossomos , Fatores de Transcrição/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...