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1.
Biochim Biophys Acta Bioenerg ; 1861(2): 148120, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734194

RESUMO

The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Ficobilissomas/química , Espectrometria de Fluorescência
2.
Anal Chem ; 92(1): 1565-1573, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31790200

RESUMO

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies.

3.
J Biol Chem ; 294(36): 13327-13335, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31320477

RESUMO

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.


Assuntos
Bordetella bronchiseptica/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Água/metabolismo , Zinco/metabolismo , Transporte Biológico , Bordetella bronchiseptica/química , Proteínas de Transporte de Cátions/química , Difusão , Água/química , Zinco/química
4.
J Biol Chem ; 294(22): 8848-8860, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30979724

RESUMO

In cyanobacterial photoprotection, the orange carotenoid protein (OCP) is photoactivated under excess light conditions and binds to the light-harvesting antenna, triggering the dissipation of captured light energy. In low light, the OCP relaxes to the native state, a process that is accelerated in the presence of fluorescence recovery protein (FRP). Despite the importance of the OCP in photoprotection, the precise mechanism of photoactivation by this protein is not well-understood. Using time-resolved X-ray-mediated in situ hydroxyl radical labeling, we probed real-time solvent accessibility (SA) changes at key OCP residues during photoactivation and relaxation. We observed a biphasic photoactivation process in which carotenoid migration preceded domain dissociation. We also observed a multiphasic relaxation process, with collapsed domain association preceding the final conformational rearrangement of the carotenoid. Using steady-state hydroxyl radical labeling, we identified sites of interaction between the FRP and OCP. In combination, the findings in this study provide molecular-level insights into the factors driving structural changes during OCP-mediated photoprotection in cyanobacteria, and furnish a basis for understanding the physiological relevance of the FRP-mediated relaxation process.


Assuntos
Proteínas de Bactérias/metabolismo , Carotenoides/metabolismo , Proteínas de Bactérias/química , Carotenoides/química , Cianobactérias/metabolismo , Radical Hidroxila/química , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Raios X
5.
Protein Pept Lett ; 26(1): 70-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30484401

RESUMO

BACKGROUND: Synchrotron hydroxyl radical footprinting is a relatively new structural method used to investigate structural features and conformational changes of nucleic acids and proteins in the solution state. It was originally developed at the National Synchrotron Light Source at Brookhaven National Laboratory in the late nineties, and more recently, has been established at the Advanced Light Source at Lawrence Berkeley National Laboratory. The instrumentation for this method is an active area of development, and includes methods to increase dose to the samples while implementing high-throughput sample delivery methods. CONCLUSION: Improving instrumentation to irradiate biological samples in real time using a sample droplet generator and inline fluorescence monitoring to rapidly determine dose response curves for samples will significantly increase the range of biological problems that can be investigated using synchrotron hydroxyl radical footprinting.


Assuntos
Cristalografia por Raios X , Radical Hidroxila , Síncrotrons , Cristalografia por Raios X/instrumentação , Cristalografia por Raios X/métodos , Cristalografia por Raios X/tendências , Corantes Fluorescentes/química , Radical Hidroxila/análise , Radical Hidroxila/química , Técnicas Analíticas Microfluídicas , Conformação Proteica , Proteínas/química
6.
Plant Physiol ; 179(1): 156-167, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389783

RESUMO

Bacterial microcompartments (BMCs) encapsulate enzymes within a selectively permeable, proteinaceous shell. Carboxysomes are BMCs containing ribulose-1,5-bisphosphate carboxylase oxygenase and carbonic anhydrase that enhance carbon dioxide fixation. The carboxysome shell consists of three structurally characterized protein types, each named after the oligomer they form: BMC-H (hexamer), BMC-P (pentamer), and BMC-T (trimer). These three protein types form cyclic homooligomers with pores at the center of symmetry that enable metabolite transport across the shell. Carboxysome shells contain multiple BMC-H paralogs, each with distinctly conserved residues surrounding the pore, which are assumed to be associated with specific metabolites. We studied the regulation of ß-carboxysome shell composition by investigating the BMC-H genes ccmK3 and ccmK4 situated in a locus remote from other carboxysome genes. We made single and double deletion mutants of ccmK3 and ccmK4 in Synechococcus elongatus PCC7942 and show that, unlike CcmK3, CcmK4 is necessary for optimal growth. In contrast to other CcmK proteins, CcmK3 does not form homohexamers; instead CcmK3 forms heterohexamers with CcmK4 with a 1:2 stoichiometry. The CcmK3-CcmK4 heterohexamers form stacked dodecamers in a pH-dependent manner. Our results indicate that CcmK3-CcmK4 heterohexamers potentially expand the range of permeability properties of metabolite channels in carboxysome shells. Moreover, the observed facultative formation of dodecamers in solution suggests that carboxysome shell permeability may be dynamically attenuated by "capping" facet-embedded hexamers with a second hexamer. Because ß-carboxysomes are obligately expressed, heterohexamer formation and capping could provide a rapid and reversible means to alter metabolite flux across the shell in response to environmental/growth conditions.


Assuntos
Proteínas de Bactérias/fisiologia , Synechococcus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Deleção de Genes , Modelos Moleculares , Simulação de Dinâmica Molecular , Permeabilidade , Synechococcus/genética
7.
J Am Chem Soc ; 139(36): 12647-12654, 2017 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-28806874

RESUMO

Achieving fast electron transfer between a material and protein is a long-standing challenge confronting applications in bioelectronics, bioelectrocatalysis, and optobioelectronics. Interestingly, naturally occurring extracellular electron transfer proteins bind to and reduce metal oxides fast enough to enable cell growth, and thus could offer insight into solving this coupling problem. While structures of several extracellular electron transfer proteins are known, an understanding of how these proteins bind to their metal oxide substrates has remained elusive because this abiotic-biotic interface is inaccessible to traditional structural methods. Here, we use advanced footprinting techniques to investigate binding between the Shewanella oneidensis MR-1 extracellular electron transfer protein MtrF and one of its substrates, α-Fe2O3 nanoparticles, at the molecular level. We find that MtrF binds α-Fe2O3 specifically, but not tightly. Nanoparticle binding does not induce significant conformational changes in MtrF, but instead protects specific residues on the face of MtrF likely to be involved in electron transfer. Surprisingly, these residues are separated in primary sequence, but cluster into a small 3D putative binding site. This binding site is located near a local pocket of positive charge that is complementary to the negatively charged α-Fe2O3 surface, and mutational analysis indicates that electrostatic interactions in this 3D pocket modulate MtrF-nanoparticle binding. Strikingly, these results show that binding of MtrF to α-Fe2O3 follows a strategy to connect proteins to materials that resembles the binding between donor-acceptor electron transfer proteins. Thus, by developing a new methodology to probe protein-nanoparticle binding at the molecular level, this work reveals one of nature's strategies for achieving fast, efficient electron transfer between proteins and materials.

8.
Nat Chem ; 9(9): 843-849, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28837177

RESUMO

Berkelium (Bk) has been predicted to be the only transplutonium element able to exhibit both +III and +IV oxidation states in solution, but evidence of a stable oxidized Bk chelate has so far remained elusive. Here we describe the stabilization of the heaviest 4+ ion of the periodic table, under mild aqueous conditions, using a siderophore derivative. The resulting Bk(IV) complex exhibits luminescence via sensitization through an intramolecular antenna effect. This neutral Bk(IV) coordination compound is not sequestered by the protein siderocalin-a mammalian metal transporter-in contrast to the negatively charged species obtained with neighbouring trivalent actinides americium, curium and californium (Cf). The corresponding Cf(III)-ligand-protein ternary adduct was characterized by X-ray diffraction analysis. Combined with theoretical predictions, these data add significant insight to the field of transplutonium chemistry, and may lead to innovative Bk separation and purification processes.

9.
Sci Rep ; 7(1): 3673, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623285

RESUMO

The human chaperonin TRiC consists of eight non-identical subunits, and its protein-folding activity is critical for cellular health. Misfolded proteins are associated with many human diseases, such as amyloid diseases, cancer, and neuropathies, making TRiC a potential therapeutic target. A detailed structural understanding of its ATP-dependent folding mechanism and substrate recognition is therefore of great importance. Of particular health-related interest is the mutation Histidine 147 to Arginine (H147R) in human TRiC subunit 5 (CCT5), which has been associated with hereditary sensory neuropathy. In this paper, we describe the crystal structures of CCT5 and the CCT5-H147R mutant, which provide important structural information for this vital protein-folding machine in humans. This first X-ray crystallographic study of a single human CCT subunit in the context of a hexadecameric complex can be expanded in the future to the other 7 subunits that form the TRiC complex.


Assuntos
Chaperonina com TCP-1/química , Chaperonina com TCP-1/genética , Suscetibilidade a Doenças , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Relação Estrutura-Atividade
10.
Inorg Chem ; 55(22): 11930-11936, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27802058

RESUMO

Targeted α therapy holds tremendous potential as a cancer treatment: it offers the possibility of delivering a highly cytotoxic dose to targeted cells while minimizing damage to surrounding healthy tissue. The metallic α-generating radioisotopes 225Ac and 227Th are promising radionuclides for therapeutic use, provided adequate chelation and targeting. Here we demonstrate a new chelating platform composed of a multidentate high-affinity oxygen-donating ligand 3,4,3-LI(CAM) bound to the mammalian protein siderocalin. Respective stability constants log ß110 = 29.65 ± 0.65, 57.26 ± 0.20, and 47.71 ± 0.08, determined for the EuIII (a lanthanide surrogate for AcIII), ZrIV, and ThIV complexes of 3,4,3-LI(CAM) through spectrophotometric titrations, reveal this ligand to be one of the most powerful chelators for both trivalent and tetravalent metal ions at physiological pH. The resulting metal-ligand complexes are also recognized with extremely high affinity by the siderophore-binding protein siderocalin, with dissociation constants below 40 nM and tight electrostatic interactions, as evidenced by X-ray structures of the protein:ligand:metal adducts with ZrIV and ThIV. Finally, differences in biodistribution profiles between free and siderocalin-bound 238PuIV-3,4,3-LI(CAM) complexes confirm in vivo stability of the protein construct. The siderocalin:3,4,3-LI(CAM) assembly can therefore serve as a "lock" to consolidate binding to the therapeutic 225Ac and 227Th isotopes or to the positron emission tomography emitter 89Zr, independent of metal valence state.


Assuntos
Quelantes/química , Complexos de Coordenação/química , Proteínas/química , Radioterapia/métodos , Tório/química , Zircônio/química , Animais , Complexos de Coordenação/farmacocinética , Feminino , Ligantes , Camundongos , Modelos Químicos , Distribuição Tecidual
11.
J Synchrotron Radiat ; 23(Pt 5): 1056-69, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27577756

RESUMO

The vast majority of biomolecular processes are controlled or facilitated by water interactions. In enzymes, regulatory proteins, membrane-bound receptors and ion-channels, water bound to functionally important residues creates hydrogen-bonding networks that underlie the mechanism of action of the macromolecule. High-resolution X-ray structures are often difficult to obtain with many of these classes of proteins because sample conditions, such as the necessity of detergents, often impede crystallization. Other biophysical techniques such as neutron scattering, nuclear magnetic resonance and Fourier transform infrared spectroscopy are useful for studying internal water, though each has its own advantages and drawbacks, and often a hybrid approach is required to address important biological problems associated with protein-water interactions. One major area requiring more investigation is the study of bound water molecules which reside in cavities and channels and which are often involved in both the structural and functional aspects of receptor, transporter and ion channel proteins. In recent years, significant progress has been made in synchrotron-based radiolytic labeling and mass spectroscopy techniques for both the identification of bound waters and for characterizing the role of water in protein conformational changes at a high degree of spatial and temporal resolution. Here the latest developments and future capabilities of this method for investigating water-protein interactions and its synergy with other synchrotron-based methods are discussed.


Assuntos
Proteínas/análise , Espectrometria de Massas , Conformação Proteica , Radiografia , Síncrotrons , Água , Raios X
12.
Methods ; 103: 49-56, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27016143

RESUMO

The assembly of the Escherichia coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.


Assuntos
Radical Hidroxila/química , RNA Bacteriano/ultraestrutura , RNA Ribossômico/ultraestrutura , Ribossomos/ultraestrutura , Ésteres do Ácido Sulfúrico/química , Técnicas de Cultura de Células , Escherichia coli , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Ribossômico/química , Ribossomos/química , Coloração e Rotulagem
13.
Proc Natl Acad Sci U S A ; 112(41): E5567-74, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26385969

RESUMO

Photoprotective mechanisms are of fundamental importance for the survival of photosynthetic organisms. In cyanobacteria, the orange carotenoid protein (OCP), when activated by intense blue light, binds to the light-harvesting antenna and triggers the dissipation of excess captured light energy. Using a combination of small angle X-ray scattering (SAXS), X-ray hydroxyl radical footprinting, circular dichroism, and H/D exchange mass spectrometry, we identified both the local and global structural changes in the OCP upon photoactivation. SAXS and H/D exchange data showed that global tertiary structural changes, including complete domain dissociation, occur upon photoactivation, but with alteration of secondary structure confined to only the N terminus of the OCP. Microsecond radiolytic labeling identified rearrangement of the H-bonding network associated with conserved residues and structural water molecules. Collectively, these data provide experimental evidence for an ensemble of local and global structural changes, upon activation of the OCP, that are essential for photoprotection.


Assuntos
Proteínas de Bactérias/química , Modelos Moleculares , Synechocystis/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
Proc Natl Acad Sci U S A ; 112(33): 10342-7, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26240330

RESUMO

Synthetic radionuclides, such as the transuranic actinides plutonium, americium, and curium, present severe health threats as contaminants, and understanding the scope of the biochemical interactions involved in actinide transport is instrumental in managing human contamination. Here we show that siderocalin, a mammalian siderophore-binding protein from the lipocalin family, specifically binds lanthanide and actinide complexes through molecular recognition of the ligands chelating the metal ions. Using crystallography, we structurally characterized the resulting siderocalin-transuranic actinide complexes, providing unprecedented insights into the biological coordination of heavy radioelements. In controlled in vitro assays, we found that intracellular plutonium uptake can occur through siderocalin-mediated endocytosis. We also demonstrated that siderocalin can act as a synergistic antenna to sensitize the luminescence of trivalent lanthanide and actinide ions in ternary protein-ligand complexes, dramatically increasing the brightness and efficiency of intramolecular energy transfer processes that give rise to metal luminescence. Our results identify siderocalin as a potential player in the biological trafficking of f elements, but through a secondary ligand-based metal sequestration mechanism. Beyond elucidating contamination pathways, this work is a starting point for the design of two-stage biomimetic platforms for photoluminescence, separation, and transport applications.


Assuntos
Elementos da Série Actinoide/química , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Proteínas/química , Elementos da Série Actinoide/farmacocinética , Quelantes/química , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Íons , Cinética , Elementos da Série dos Lantanídeos , Ligantes , Luminescência , Metais/química , Conformação Molecular , Centrais Nucleares , Fotoquímica , Ligação Proteica , Liberação Nociva de Radioativos , Espectrofotometria , Eletricidade Estática , Difração de Raios X
15.
EMBO J ; 31(3): 731-40, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22193720

RESUMO

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.


Assuntos
Nucleotídeos de Adenina/metabolismo , Chaperoninas do Grupo II/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Chaperoninas do Grupo II/química , Hidrólise , Cinética , Modelos Moleculares , Conformação Proteica
16.
Structure ; 18(11): 1471-82, 2010 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-21070946

RESUMO

The major cAMP receptors in eukaryotes are the regulatory (R) subunits of PKA, an allosteric enzyme conserved in fungi through mammals. While mammals have four R-subunit genes, Saccharomyces cerevisiae has only one, Bcy1. To achieve a molecular understanding of PKA activation in yeast and to explore the evolution of cyclic-nucleotide binding (CNB) domains, we solved the structure of cAMP-bound Bcy1(168-416). Surprisingly, the relative orientation of the two CNB domains in Bcy1 is very different from mammalian R-subunits. This quaternary structure is defined primarily by a fungi-specific sequence in the hinge between the αB/αC helices of the CNB-A domain. The unique interface between the two CNB domains in Bcy1 defines the allosteric mechanism for cooperative activation of PKA by cAMP. Some interface motifs are isoform-specific while others, although conserved, play surprisingly different roles in each R-subunit. Phylogenetic analysis shows that structural differences in Bcy1 are shared by fungi of the subphylum Saccharomycotina.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Evolução Molecular , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química , Sequências Reguladoras de Ácido Nucleico/genética , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais/fisiologia , Análise por Conglomerados , Cristalografia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Simulação de Dinâmica Molecular , Filogenia , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais/genética
17.
J Biol Chem ; 285(36): 27958-66, 2010 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-20573955

RESUMO

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.


Assuntos
Chaperoninas do Grupo II/química , Dobramento de Proteína , Trifosfato de Adenosina/metabolismo , Cristalografia por Raios X , Chaperoninas do Grupo II/metabolismo , Hidrólise , Mathanococcus , Modelos Moleculares , Estrutura Terciária de Proteína
18.
Biochemistry ; 41(18): 5799-806, 2002 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-11980483

RESUMO

We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated DeltaDeltaG values of >2 kcal x mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge. The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present. This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.


Assuntos
Cátions Bivalentes/metabolismo , Cátions Monovalentes/metabolismo , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/metabolismo , Tetrahymena/enzimologia , Tetrahymena/genética , Animais , Sequência de Bases , Catálise , Cátions Bivalentes/farmacologia , Cátions Monovalentes/farmacologia , Magnésio/metabolismo , Magnésio/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Potássio/metabolismo , Potássio/farmacologia , RNA Catalítico/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Sódio/metabolismo , Sódio/farmacologia , Termodinâmica , Ureia/farmacologia
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