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1.
J Agric Food Chem ; 67(23): 6633-6641, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31099573

RESUMO

Ferritin is a multisubunit protein with a hollow interior interface and modifiable surfaces. In this study, the manothermosonication (MTS) technology was applied to apo-red bean seed ferritin (apoRBF) to produce the MTS-treated apoRBF (MTFS). MTS treatment (200 kPa, 50 °C, and 40 s) maintained the spherical morphology of apoRBF (12 nm), but reduced the content of α-helix structure and increased the content of random coil structure, and correspondingly decreased the ferritin stability. The MTS treatment also affected the ferritin's iron storage function by decreasing its iron oxidative deposition activity and increasing the iron release activity. Importantly, the disassembly and reassembly properties of the MTFS induced by pH changes were retained, which facilitated its usage in encapsulation of tea polyphenol-epigallocatechin gallate (EGCG) into the ferritin by a relatively benign pH conversion routine (pH 3.0/6.8). In addition, the water solubility of the MTFS was increased, leading to the improved encapsulation efficiency of the EGCG molecules. This study will facilitate the ferritin modification and functionalization by MTS to design a protein variant to be used as new scaffold for iron and bioactive compounds.


Assuntos
Apoferritinas/química , Apoproteínas/química , Portadores de Fármacos/química , Fabaceae/química , Ferro/química , Proteínas de Plantas/química , Sonicação/métodos , Apoferritinas/isolamento & purificação , Apoproteínas/isolamento & purificação , Catequina/análogos & derivados , Catequina/química , Portadores de Fármacos/isolamento & purificação , Concentração de Íons de Hidrogênio , Oxirredução , Proteínas de Plantas/isolamento & purificação , Estabilidade Proteica , Solubilidade , Sonicação/instrumentação
2.
Vopr Pitan ; 88(1): 91-96, 2019.
Artigo em Russo | MEDLINE | ID: mdl-30811139

RESUMO

In this work, studies were carried out to obtain and determine the iron-binding ability of lactoferrin isolated from milk of Holstein-Friesian (black-and-white) breed of cows, which is the main stock of the Russian cattle herd (CH). Aim of the study was to obtain lactoferrin and determine its iron-binding capacity for substantiating the raw material resources of its industrial production as an easily digestible source of ferrous iron for the production of dietary supplements and/or specialized foods. MATERIAL AND METHODS: To optimize the production of lactoferrin in the conditions of dairy enterprises, we used a method of lactoferrin isolation from cow's milk in its own modification, which consisted in the degreasing of whole milk by centrifugation and double cation-exchange chromatography with successive application of the following sorbents: CM-cellulose (CM-52) and Macro-Prep High Q Support. RESULTS AND DISCUSSION: The developed modification of the method of chromatographic production of lactoferrin has shown its effectiveness and availability for production at domestic dairy enterprises. The purity of lactoferrin is about 95%, and the content is about 74 µg/cm3. Iron-binding capacity was determined in apo- and holoforms of lactoferrin. The ability of saturation of apolactoferrin with iron has been shown. CONCLUSION: The new obtained factual material allows us to express the prerequisites for further research to justify the possibility of using the iron-saturated form of hololactoferrin of cow milk of the Holstein-Frisian breed as a domestic raw material for dietary supplements and specialized foods.


Assuntos
Apoproteínas/química , Apoproteínas/isolamento & purificação , Ferro/química , Lactoferrina/química , Lactoferrina/isolamento & purificação , Leite/química , Animais , Bovinos
3.
J Biol Chem ; 292(33): 13658-13671, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28596380

RESUMO

Hemopexin protects against heme toxicity in hemolytic diseases and conditions, sepsis, and sickle cell disease. This protection is sustained by heme-hemopexin complexes in biological fluids that resist oxidative damage during heme-driven inflammation. However, apo-hemopexin is vulnerable to inactivation by reactive nitrogen (RNS) and oxygen species (ROS) that covalently modify amino acids. The resultant nitration of amino acids is considered a specific effect reflecting biological events. Using LC-MS, we discovered low endogenous levels of tyrosine nitration in the peptide YYCFQGNQFLR in the heme-binding site of human hemopexin, which was similarly nitrated in rabbit and rat hemopexins. Immunoblotting and selective reaction monitoring were used to quantify tyrosine nitration of in vivo samples and when hemopexin was incubated in vitro with nitrating nitrite/myeloperoxidase/glucose oxidase. Significantly, heme binding by hemopexin declined as tyrosine nitration proceeded in vitro Three nitrated tyrosines reside in the heme-binding site of hemopexin, and we found that one, Tyr-199, interacts directly with the heme ring D propionate. Investigating the oxidative modifications of amino acids after incubation with tert-butyl hydroperoxide and hypochlorous acid in vitro, we identified additional covalent oxidative modifications on four tyrosine residues and one tryptophan residue of hemopexin. Importantly, three of the four modified tyrosines, some of which have more than one modification, cluster in the heme-binding site, supporting a hierarchy of vulnerable amino acids. We propose that during inflammation, apo-hemopexin is nitrated and oxidated in niches of the body containing activated RNS- and ROS-generating immune and endothelial cells, potentially impairing hemopexin's protective extracellular antioxidant function.


Assuntos
Hemopexina/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Animais , Apoproteínas/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Heme/química , Heme/metabolismo , Hemopexina/química , Hemopexina/isolamento & purificação , Humanos , Cinética , Ligantes , Estrutura Molecular , Oxirredução , Conformação Proteica , Coelhos , Ratos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Espectrometria de Massas em Tandem , Triptofano/química , Tirosina/química
4.
J Mol Model ; 20(9): 2400, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25152065

RESUMO

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.


Assuntos
Apoproteínas/química , Proteínas de Escherichia coli/química , Metionina/análogos & derivados , Simulação de Dinâmica Molecular , Proteínas Repressoras/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/metabolismo , Cinética , Metionina/química , Metionina/metabolismo , Oxirredução , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
5.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 1): 87-91, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24419625

RESUMO

Different genome-editing strategies have fuelled the development of new DNA-targeting molecular tools allowing precise gene modifications. Here, the expression, purification, crystallization and preliminary X-ray diffraction of BurrH, a novel DNA-binding protein from Burkholderia rhizoxinica, are reported. Crystallization experiments of BurrH in its apo form and in complex with its target DNA yielded crystals suitable for X-ray diffraction analysis. The crystals of the apo form belonged to the primitive hexagonal space group P3(1) or its enantiomorph P3(2), with unit-cell parameters a = b = 73.28, c = 268.02 Å, α = ß = 90, γ = 120°. The BurrH-DNA complex crystallized in the monoclinic space group P2(1), with unit-cell parameters a = 70.15, b = 95.83, c = 76.41 Å, α = γ = 90, ß = 109.51°. The self-rotation function and the Matthews coefficient suggested the presence of two protein molecules per asymmetric unit in the apo crystals and one protein-DNA complex in the monoclinic crystals. The crystals diffracted to resolution limits of 2.21 and 2.65 Å, respectively, using synchrotron radiation.


Assuntos
Apoproteínas/química , Proteínas de Bactérias/química , Burkholderia/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/isolamento & purificação , DNA/química , Difração de Raios X , Apoproteínas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Cristalização , DNA/metabolismo
6.
Biochemistry ; 53(2): 311-22, 2014 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-24364624

RESUMO

Bacterial receiver domains modulate intracellular responses to external stimuli in two-component systems. Sma0114 is the first structurally characterized representative from the family of receiver domains that are substrates for histidine-tryptophan-glutamate (HWE) kinases. We report the NMR structure of Sma0114 bound by Ca(2+) and BeF3(-), a phosphate analogue that stabilizes the activated state. Differences between the NMR structures of the inactive and activated states occur in helix α1, the active site loop that connects strand ß3 and helix α3, and in the segment from strand ß5 to helix α5 of the 455 (α4-ß5-α5) face. Structural rearrangements of the 455 face typically make receiver domains competent for binding downstream target molecules. In Sma0114 the structural changes accompanying activation result in a more negatively charged surface for the 455 face. Coupling between the 455 face and active site phosphorylation is usually mediated through the rearrangement of a threonine and tyrosine residue, in a mechanism called Y-T coupling. The NMR structure indicates that Sma0114 lacks Y-T coupling and that communication between the active site and the 455 face is achieved through a conserved lysine residue that stabilizes the acyl phosphate in receiver domains. (15)N-NMR relaxation experiments were used to investigate the backbone dynamics of the Sma0114 apoprotein, the binary Sma0114·Ca(2+) complex, and the ternary Sma0114·Ca(2+)·BeF3(-) complex. The loss of entropy due to ligand binding at the active site is compensated by increased flexibility in the 455 face. The dynamic character of the 455 face in Sma0114, which results in part from the replacement of helix α4 by a flexible loop, may facilitate induced-fit recognition of target molecules.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Quinases/metabolismo , Sinorhizobium meliloti/química , Apoproteínas/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Proteínas de Bactérias/isolamento & purificação , Berílio/química , Berílio/metabolismo , Cálcio/química , Cálcio/metabolismo , Domínio Catalítico , Fluoretos/química , Fluoretos/metabolismo , Histidina Quinase , Modelos Moleculares , Conformação Proteica , Proteínas Quinases/química
7.
Biometals ; 26(6): 887-96, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23917727

RESUMO

The stoichiometric analysis of the metal induced Metallothionein (MT) is pertinent for understanding the metal-MT interactions. Despite innumerable publications on MT, the literature addressing these aspects is limited. To bridge this gap, PIXE and ESI-MS analysis of the commercial rabbit liver MT1 (an isoform of MT), zinc induced isolated rat liver MT1, apo and Arsenic substituted rabbit liver MT1 have been carried out. These techniques in combination provide information about number and the signature of all the metal ions bound to MT. By using ESI-MS in the rabbit MT1, ions of Zn n MT1 (n = 0, 1, 4, 5, 6, 7) whereas, in rat MT1, the Zn1MT1 and Zn5MT1 ions are observed. PIXE analysis shows that some copper along with zinc is also present in the rabbit as well as rat MT1 which could not be assessed with ESI-MS. During As metallation reaction with rabbit MT1, with increase in arsenic concentration, the amount of arsenic bound to MT1 also increases, though not proportionally. The presence of both Zn and Cu in MT1 on Zn supplementation can be related to the role of MT in Zn and Cu homeostasis. Further, the presence of partially metallated MT1 suggests that MT1 may donate fractional amount of metal from it's fully metallated form to other proteins where Zn acts as a cofactor.


Assuntos
Apoproteínas/química , Arsênico/química , Cobre/química , Metalotioneína/química , Zinco/química , Animais , Apoproteínas/isolamento & purificação , Sítios de Ligação , Fígado/química , Fígado/metabolismo , Masculino , Metalotioneína/isolamento & purificação , Ligação Proteica , Coelhos , Ratos , Ratos Wistar , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Espectrometria por Raios X
8.
Methods Mol Biol ; 963: 87-97, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23296606

RESUMO

X-ray crystallography is a potent and meanwhile fast technique to obtain detailed structural information of S100 proteins in their apo or metal ion-loaded state. S100 proteins crystallize in the absence or presence of Ca(2+) and Zn(2+) and the obtained crystals often diffract to high resolution yielding information on the ion-binding sites, conformation, and target interaction sites of the proteins. Here, I describe a general scheme to isolate and crystallize S100 proteins and the analysis of protein crystals using a modern synchrotron source.


Assuntos
Cristalografia por Raios X/métodos , Proteínas S100/química , Motivos de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Cristalografia por Raios X/instrumentação , Escherichia coli/genética , Modelos Moleculares , Proteínas S100/genética , Proteínas S100/isolamento & purificação , Síncrotrons
9.
Methods Mol Biol ; 849: 373-86, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22528103

RESUMO

The S100 proteins are a large family of 10-12 kDa EF-hand signaling proteins that bind calcium, and in some cases zinc and copper, functioning as central regulators in a diversity of cellular processes. These proteins have tissue, cell, and subcellular-specific expression patterns, and many have an extracellular function. Altogether, these properties underlie their functional diversity and involvement in several pathological conditions including cancer, inflammation, and neurodegeneration. S100 proteins exhibit considerable structural plasticity, being able to exist as monomers or assemble into dimers, higher oligomers, and amyloids, frequently in a metal-dependent manner. Many of these oligomers are functionally relevant, and S100 amyloids have been recently found in prostatic inclusions. Here, we report experimental procedures for the isolation and quantitation of S100 oligomers from tissues, purification of recombinant human S100 protein for assays and use as standards, and an amyloidogenesis assay that allows monitoring the formation of S100 ß-oligomers and amyloids in apo- and metal-bound S100 proteins.


Assuntos
Amiloide/química , Multimerização Proteica , Proteínas S100/química , Amiloide/genética , Amiloide/isolamento & purificação , Amiloide/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Benzotiazóis , Encéfalo/citologia , Humanos , Luz , Metais/metabolismo , Estrutura Secundária de Proteína , Proteínas S100/genética , Proteínas S100/isolamento & purificação , Proteínas S100/metabolismo , Espalhamento de Radiação , Espectroscopia de Infravermelho com Transformada de Fourier , Tiazóis/metabolismo
10.
Biophys J ; 100(11): 2773-82, 2011 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-21641323

RESUMO

Expanded CAG diseases are progressive neurodegenerative disorders in which specific proteins have an unusually long polyglutamine stretch. Although these proteins share no other sequence or structural homologies, they all aggregate into intracellular inclusions that are believed to be pathological. We sought to determine what impact the position and number of glutamines have on the structure and aggregation of the host protein, apomyoglobin. Variable-length polyQ tracts were inserted either into the loop between the C- and D-helices (Q(n)CD) or at the N-terminus (Q(n)NT). The Q(n)CD mutants lost some α-helix and gained unordered and/or ß-sheet in a length-dependent manner. These mutants were partially unfolded and rapidly assembled into soluble chain-like oligomers. In sharp contrast, the Q(n)NT mutants largely retained wild-type tertiary structure but associated into long, fibrillar aggregates. Control proteins with glycine-serine repeats (GS(8)CD and GS(8)NT) were produced. GS(8)CD exhibited similar structural perturbations and aggregation characteristics to an analogously sized Q(16)CD, indicating that the observed effects are independent of amino acid composition. In contrast to Q(16)NT, GS(8)NT did not form fibrillar aggregates. Thus, soluble oligomers are produced through structural perturbation and do not require polyQ, whereas classic fibrils arise from specific polyQ intermolecular interactions in the absence of misfolding.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Mioglobina/química , Mioglobina/metabolismo , Peptídeos/metabolismo , Dobramento de Proteína , Multimerização Proteica , Apoproteínas/genética , Apoproteínas/isolamento & purificação , Cinética , Modelos Moleculares , Mutação , Mioglobina/genética , Mioglobina/isolamento & purificação , Conformação Proteica , Redobramento de Proteína , Estabilidade Proteica
11.
Biochem Cell Biol ; 89(3): 299-307, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21539457

RESUMO

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.


Assuntos
Aminoácido Oxirredutases/isolamento & purificação , Apoproteínas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Di-Hidrolipoamida Desidrogenase/isolamento & purificação , Escherichia coli/metabolismo , Complexos Multienzimáticos/isolamento & purificação , Peptídeo Sintases/isolamento & purificação , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Transferases/isolamento & purificação , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Sequência de Aminoácidos , Apoproteínas/biossíntese , Apoproteínas/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Cromatografia de Afinidade , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/genética , Di-Hidrolipoamida Desidrogenase/biossíntese , Di-Hidrolipoamida Desidrogenase/genética , Escherichia coli/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Histidina/metabolismo , Humanos , Hiperglicinemia não Cetótica/enzimologia , Hiperglicinemia não Cetótica/patologia , Dados de Sequência Molecular , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Oligopeptídeos/metabolismo , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Pichia/genética , Cultura Primária de Células , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência , Pele/citologia , Pele/enzimologia , Transferases/biossíntese , Transferases/genética
12.
Biotechnol Appl Biochem ; 57(3): 87-95, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20923415

RESUMO

We investigated and optimized a purification process, suitable for industrial scale, to obtain pharmaceutical grade apo-Tf (apo-transferrin), preserving its physiological properties and functions. Apo-Tf was obtained from fraction IV subfraction 1 and IV subfraction 4 (fraction IV-1,4), a waste product of the Cohn fractionation process, performing a single chromatographic run and two viral inactivation/removal steps. The structural integrity and the biological activity of the final product were extensively tested. The yield of apo-Tf produced was 80% on laboratory scale and 90% in scale-up lots, and the purity was higher than 95%. The purified protein preserves iron- and receptor-binding activities and shows a normal glycosylation pattern. The single chromatographic step process presented here provides an efficient means to prepare commercial quantities of the protein. The final product is sterile and two viral inactivation/removal steps were introduced into the process.


Assuntos
Apoproteínas/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Fracionamento Químico/métodos , Cromatografia por Troca Iônica/métodos , Transferrina/isolamento & purificação , Apoproteínas/metabolismo , Proteínas Sanguíneas/metabolismo , Proliferação de Células , Cromatografia por Troca Iônica/economia , Células HeLa , Humanos , Ferro/metabolismo , Ligação Proteica , Estabilidade Proteica , Transferrina/metabolismo
13.
Methods Mol Biol ; 652: 341-61, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20552439

RESUMO

STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol-binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely expressed in diverse adult organs and throughout embryonic development. Mutations in human STRA6 that abolish its vitamin A uptake activity cause severe pathological phenotypes in many human organs including the eye, brain, lung, and heart. This chapter describes functional assays for STRA6 in live cells and on cellular membranes. These assays can be employed to study the mechanism of this new membrane transport mechanism and its roles in the physiology and pathology of many organs.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Vitamina A/metabolismo , Animais , Apoproteínas/biossíntese , Apoproteínas/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Transporte Biológico , Células COS , Membrana Celular/metabolismo , Sobrevivência Celular , Sistema Livre de Células/metabolismo , Ésteres , Humanos , Proteínas de Membrana/química , Imagem Molecular , Renaturação Proteica , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Ligação ao Retinol/biossíntese , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/isolamento & purificação , Proteínas de Ligação ao Retinol/metabolismo , Solubilidade , Trítio/química
14.
Biochem Biophys Res Commun ; 386(4): 617-22, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19549504

RESUMO

Blue fluorescent protein of aequorin (BFP) is a complex of Ca(2+)-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin.


Assuntos
Equorina/biossíntese , Equorina/química , Apoproteínas/biossíntese , Apoproteínas/química , Benzenoacetamidas/química , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/química , Pirazinas/química , Equorina/isolamento & purificação , Apoproteínas/isolamento & purificação , Benzenoacetamidas/síntese química , Escherichia coli/metabolismo , Proteínas Luminescentes/isolamento & purificação , Pirazinas/síntese química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência
15.
Bioelectrochemistry ; 75(2): 182-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19427819

RESUMO

Ruthenium (II) complex of mesoporphyrin-IX was incorporated into apomyoglobin to prepare artificial ruthenium myoglobin (RuMb) containing the ruthenium porphyrin in place of protoheme. The electrochemical and spectral characteristics (i.e., UV and CD spectra) of RuMb were investigated in comparison with wild type myoglobin. The effect of the metal center on the redox properties of myoglobin is directly observed by electrochemical analysis, all of which may be compared with similar measurements of the wild type myoglobin. Unlike other metal reconstituted myoglobins, i.e., cobalt myoglobin and manganese myoglobin, fast and reversible electron transfer properties were observed for RuMb, which is comparable with wild type myoglobin. The formal potential of 170 (+/-10) mV vs. Ag|AgCl (sat. KCl) of RuMb was directly determined for the first time by cyclic voltammetry, where the k(0)' value was estimated to be about 3(+/-0.2)x10(-4) cm s(-1) at pH 6.8. Mediatorless and reversible spectroelectrochemical behaviors were also observed using an optically transparent thin-layer electrode cell (OTTLE). The present results suggest that the major redox properties of the protein result from both the metal porphyrin center and globin environment. The novel redox properties predict that the engineered RuMb has analogous biofunctionalities to the wild type myoglobin in contrast to other metal reconstituted myoglobins.


Assuntos
Mioglobina/química , Rutênio/química , Animais , Apoproteínas/química , Apoproteínas/isolamento & purificação , Eletroquímica , Transporte de Elétrons , Cavalos , Estrutura Molecular , Miocárdio/química , Mioglobina/isolamento & purificação , Oxirredução , Porfirinas/síntese química , Porfirinas/química , Engenharia de Proteínas , Análise Espectral
16.
J Inorg Biochem ; 103(3): 316-25, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19135260

RESUMO

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman's reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.


Assuntos
Heme Oxigenase-1/metabolismo , Heme/metabolismo , Ferro/metabolismo , Selenocisteína/metabolismo , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Heme Oxigenase-1/genética , Humanos , Ligantes , Plasmídeos/genética , Plasmídeos/metabolismo
17.
J Am Chem Soc ; 131(7): 2739-46, 2009 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-19170491

RESUMO

During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner.


Assuntos
Apoproteínas/química , Flavodoxina/química , Alanina/química , Substituição de Aminoácidos , Apoproteínas/isolamento & purificação , Azotobacter vinelandii/química , Cisteína/química , Flavodoxina/isolamento & purificação , Cinética , Ressonância Magnética Nuclear Biomolecular/métodos , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Termodinâmica
18.
Arch Biochem Biophys ; 479(1): 69-73, 2008 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-18782555

RESUMO

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.


Assuntos
Íons/química , Peptídeos/química , Proteínas/química , Animais , Ânions/química , Apoproteínas/isolamento & purificação , Apoproteínas/metabolismo , Cátions/química , Citocromos c/química , Desulfovibrio desulfuricans/química , Estabilidade Enzimática , Enzimas/química , Escherichia coli/genética , Flavodoxina/isolamento & purificação , Flavodoxina/metabolismo , Cavalos , Temperatura Alta , Concentração de Íons de Hidrogênio , Miocárdio/enzimologia , Desnaturação Proteica , Propriedades de Superfície , Termodinâmica , Água/química
19.
Electrophoresis ; 29(13): 2790-800, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18546173

RESUMO

Metallothioneins (MTs) are a group of low-molecular-mass proteins (6-7 kDa) characterized by their great affinity for heavy metal ions. At acidic pH, the bound metal ions are released from the amino acidic structure and MTs lead to apothioneins. In this study, a general equation is used to model the electrophoretic mobility of rabbit liver apothioneins as a function of the pH of the separation electrolyte. The ability of these relationships to explain the migration behavior of these relatively complex polyprotic proteins in the pH range between 2 and 6 has been investigated. Their relevant ionization constant values in the studied pH range were estimated and employed for molecular charge calculations. The classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (me vs. q/Malpha) were tested for prediction of their electrophoretic separations. The accuracy of the separations predicted at acidic pH was confirmed by CE-ESI-MS.


Assuntos
Apoproteínas/isolamento & purificação , Eletroforese Capilar/métodos , Fígado/química , Metalotioneína/isolamento & purificação , Sequência de Aminoácidos , Animais , Modelos Teóricos , Isoformas de Proteínas/isolamento & purificação , Coelhos , Espectrometria de Massas por Ionização por Electrospray/métodos
20.
J Mol Biol ; 380(5): 900-16, 2008 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-18579154

RESUMO

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.


Assuntos
Antígenos CD/metabolismo , Apoproteínas/metabolismo , Cobalto/metabolismo , Ferro/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Sequência de Aminoácidos , Antígenos CD/sangue , Antígenos CD/química , Antígenos CD/isolamento & purificação , Apoproteínas/sangue , Apoproteínas/química , Apoproteínas/isolamento & purificação , Sítios de Ligação , Citoplasma/metabolismo , Dimerização , Dissulfetos/química , Feminino , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Placenta/química , Gravidez , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Prótons , Receptores da Transferrina/sangue , Receptores da Transferrina/química , Receptores da Transferrina/isolamento & purificação , Espectrometria de Fluorescência , Temperatura , Termodinâmica , Transferrina/química , Transferrina/isolamento & purificação
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