RESUMO
The presented data were obtained with the lipocalin allergen Mus m 1.0102 and its cysteine mutants MM-C138A, MM-C157A and MM-C138,157A, whose structural features and unfold reversibility investigations are presented in the research article entitled "The allergen Mus m 1.0102: cysteine residues and molecular allergology" [1]. The data were obtained by means of a Dynamic Light Scattering-based thermal stability assay, a Fluorescence-based ligand-binding assay and a basophil degranulation test, and describe proteins' fold stability, ligand binding ability and allergenic potential, respectively. Analysis of the collected data produced the temperatures corresponding to the onset of the protein unfolding, the dissociation constants for N-Phenyl-1-naphthylamine ligand and the profiles of ß-hexosaminidase release from RBL SX-38 cells, sensitized with the serum of selected allergic patients and incubated with increasing antigens concentrations. These data allow for comparison of the lipocalin allergen Mus m 1.0102 with its conserved cysteines mutants and, with regard to their potential application in allergy diagnostics and immunotherapy, they contribute to the process of recombinant allergen characterization and standardization.
RESUMO
Mus m 1.0102 is a member of the mouse Major Urinary Protein family, belonging to the Lipocalins superfamily. Major Urinary Proteins (MUPs) are characterized by highly conserved structural motifs. These include a disulphide bond, involved in protein oxidative folding and protein structure stabilization, and a free cysteine residue, substituted by serine only in the pheromonal protein Darcin (MUP20). The free cysteine is recognized as responsible for the onset of inter- or intramolecular thiol/disulphide exchange, an event that favours protein aggregation. Here we show that the substitution of selected cysteine residues modulates Mus m 1.0102 protein folding, fold stability and unfolding reversibility, while maintaining its allergenic potency. Recombinant allergens used for immunotherapy or employed in allergy diagnostic kits require, as essential features, conformational stability, sample homogeneity and proper immunogenicity. In this perspective, recombinant Mus m 1.0102 might appear reasonably adequate as lead molecule because of its allergenic potential and thermal stability. However, its modest resistance to aggregation renders the protein unsuitable for pharmacological preparations. Point mutation is considered a winning strategy. We report that, among the tested mutants, C138A mutant acquires a structure more resistant to thermal stress and less prone to aggregation, two events that act positively on the protein shelf life. Those features make that MUP variant an attractive lead molecule for the development of a diagnostic kit and/or a vaccine.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Proteínas/química , Proteínas/imunologia , Alérgenos/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Cisteína/química , Humanos , Testes Imunológicos , Ligantes , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Gelonin from the Indian plant Gelonium multiflorum belongs to the type I ribosome-inactivating proteins (RIPs). Like other members of RIPs, this toxin glycoprotein inhibits protein synthesis of eukaryotic cells; hence, it is largely used in the construction of immunotoxins composed of cell-targeted antibodies. Lysosomal degradation is one of the main issues in targeted tumor therapies, especially for type I RIP-based toxins, as they lack the translocation domains. The result is an attenuated cytosolic delivery and a decrease of the antitumor efficacy of these plant-derived toxins; therefore, strategies to permit their release from endosomal vesicles or modifications of the toxins to make them resistant to degradation are necessary to improve their efficacy. Using infrared spectroscopy, we thoroughly analyzed both the secondary structure and the thermal unfolding of gelonin. Moreover, by the combination of two-dimensional correlation spectroscopy and phase diagram method, it was possible to deduce the sequence of events during the unfolding, confirming the typical characteristic of the RIP members to denature in two steps, as a sequential loss of tertiary and secondary structure was detected at 58 °C and at 65 °C, respectively. Additionally, some discrepancies in the unfolding process between gelonin and saporin-S6, another type I RIP protein, were detected.
Assuntos
Temperatura Alta , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/química , Suregada/química , Toxinas Biológicas/química , Dicroísmo Circular , Estrutura Secundária de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Sementes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Toxinas Biológicas/isolamento & purificaçãoRESUMO
Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein.
Assuntos
Proteínas Inativadoras de Ribossomos Tipo 1/química , Saponaria/química , Sementes/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Desnaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Proteínas Inativadoras de Ribossomos Tipo 1/isolamento & purificação , Saporinas , Soluções , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Thymoquinone (TQ) is the main bioactive component isolated from Nigella sativa essential oil and seeds and has been used for the treatment of inflammations, liver disorders, arthritis, and is of great importance as a promising therapeutic drug for different diseases including cancer. This paper reports the first experimental evidence on binding of TQ to human α(1)-acid glycoprotein (AGP), an important drug-binding glycoprotein in human plasma, which affects pharmacokinetic properties of various therapeutic agents. The interaction of TQ with AGP has been characterized by Fourier transform infrared (FTIR) and fluorescence spectroscopy, as well as by molecular docking experiments. FTIR spectroscopy showed that the binding of TQ to AGP slightly increases its thermal stability and shifts the existence of a molten globule-like state observed in a previous study to higher temperature. The binding constants K(a); the number of binding sites n; and the corresponding thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated through fluorescence spectroscopy. Fluorescence quenching experiments indicated that TQ binding involves hydrophobic interactions and to a lower extent hydrogen bonds, in agreement with molecular docking experiments. The data on binding ability of TQ to AGP represent basic information for the TQ pharmacokinetics such as drug metabolism and distribution in the body.
Assuntos
Benzoquinonas/farmacologia , Nigella sativa/química , Orosomucoide/metabolismo , Benzoquinonas/química , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Orosomucoide/química , Óleos de Plantas/química , Ligação Proteica , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , TermodinâmicaRESUMO
In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.
Assuntos
Antivenenos/química , Glicoproteínas/química , Mucuna/química , Peptídeos/química , Proteínas de Plantas/química , Sementes/química , Antivenenos/farmacologia , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/análise , Glicoproteínas/farmacologia , Concentração de Íons de Hidrogênio , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/farmacologia , Estabilidade Proteica , Estrutura Secundária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Fourier-transform infrared spectroscopy, in vitro bioassay and enzyme-linked immunoassay were used to study the structural-functional relationships of recombinant mink growth hormone (mGH), refolded and stored under different conditions. Porcine GH (pGH) was synthesized and used as an example. These two hormones, when refolded and stored the same way, had the same secondary structures, biological and immunological efficacy, and biological potency. Only the immunological potency differed, mGH being significantly less potent than pGH. Renaturation pH and storing frozen or at 4 degrees C in 5% glycerol did not affect either the secondary structure or the activity. However, freeze-drying raised the content of buried alpha-helices and lowered that of solvated alpha-helices and of unordered structures. These conformational changes were associated with a reduction of immunological and biological potency of mGH and of immunological potency of pGH. These findings provide original information on the secondary structure of mGH, and show that conformational changes induced by lyophilization adversely affect its activity.
Assuntos
Hormônio do Crescimento/química , Hormônio do Crescimento/imunologia , Vison/imunologia , Renaturação Proteica , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Liofilização , Hormônio do Crescimento/genética , Camundongos , Vison/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade , Suínos , TemperaturaRESUMO
The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Transporte de Monossacarídeos/química , Técnicas Biossensoriais , Cisteína/química , Corantes Fluorescentes/farmacologia , Glucose/química , Glucose/farmacologia , Ligantes , Metionina/química , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteômica/métodos , Salmonella typhimurium/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
Hsp70 (DnaK) is a highly conserved molecular chaperone present in bacteria, eukaryotes, and some archaea. In a previous work we demonstrated that DnaK from the archaeon Methanosarcina mazei (DnaK(Mm)) and the DnaK from the bacterium Escherichia coli (DnaK(Ec)) were functionally similar when assayed in vitro but DnaK(Mm) failed to substitute for DnaK(Ec) in vivo. Searching for the molecular basis of the observed DnaK species specificity we compared substrate binding by DnaK(Mm) and DnaK(Ec). DnaK(Mm) showed a lower affinity for the model peptide (a-CALLQSRLLS) compared to DnaK(Ec). Furthermore, it was unable to negatively regulate the E. coli sigma32 transcription factor level under heat shock conditions and poorly bound purified sigma32, which is a native substrate of DnaK(Ec). These observations taken together indicate differences in substrate specificity of archaeal and bacterial DnaKs. Structural modeling of DnaK(Mm) showed some structural differences in the substrate-binding domains of DnaK(Mm) and DnaK(Ec), which may be responsible, at least partially, for the differences in peptide binding. Size-exclusion chromatography and native gel electrophoresis revealed that DnaK(Mm) was found preferably in high molecular mass oligomeric forms, contrary to DnaK(Ec). Oligomers of DnaK(Mm) could be dissociated in the presence of ATP and a substrate (peptide) but not ADP, which may suggest that monomer is the active form of DnaK(Mm).
Assuntos
Proteínas Arqueais/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Methanosarcina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Sítios de Ligação , Cromatografia em Gel , Dimerização , Eletroforese em Gel de Poliacrilamida , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico , Immunoblotting , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Fator sigma/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Especificidade por SubstratoRESUMO
The effect of SDS, pD, and temperature on the structure and stability of the protein disulfide oxidoreductase from Pyrococcus furiosus (PfPDO) was investigated by molecular dynamic (MD) simulations and FT-IR spectroscopy. pD affects the thermostability of alpha-helices and beta-sheets differently, and 0.5% or higher SDS concentration influences the structure significantly. The experiments allowed us to detect a secondary structural reorganization at a definite temperature and pD which may correlate with a high ATPase activity of the protein. The MD simulations supported the infrared data and revealed the different behavior of the N and C terminal segments, as well as of the two active sites.
Assuntos
NADH NADPH Oxirredutases/química , Pyrococcus furiosus/metabolismo , Dodecilsulfato de Sódio/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sítios de Ligação , Simulação por Computador , Dissulfetos , Temperatura Alta , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas/química , Pyrococcus furiosus/enzimologia , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
HtrA heat shock protease is highly conserved in evolution, and in Escherichia coli, it protects the cell by degradation of proteins denatured by heat and oxidative stress, and also degrades misfolded proteins with reduced disulfide bonds. The mature, 48-kDa HtrA undergoes partial autocleavage with formation of two approximately 43 kDa truncated polypeptides. We showed that under reducing conditions, the HtrA level in cells was increased and efficient autocleavage occurred, while heat shock and oxidative shock caused the increase of HtrA level, but not the autocleavage. Purified HtrA cleaved itself during proteolysis of substrates but only under reducing conditions. These results indicate that the autocleavage is triggered specifically by proteolysis under reducing conditions, and is a physiological process occurring in cells. Conformations of reduced and oxidized forms of HtrA differed as judged by SDS-PAGE, indicating presence of a disulfide bridge in native protein. HtrA mutant protein lacking Cys57 and Cys69 was autocleaved even without the reducing agents, which indicates that the cysteines present in the N-terminal region are necessary for stabilization of HtrA peptide. Autocleavage caused the native, hexameric HtrA molecules dissociate into monomers that were still proteolytically active. This shows that the N-terminal part of HtrA is essential for maintaining quaternary structure of HtrA.
Assuntos
Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas Periplásmicas/química , Estrutura Quaternária de Proteína , Serina Endopeptidases/química , Sequência de Aminoácidos , Cisteína/química , Estabilidade Enzimática , Escherichia coli/química , Oxirredução , Fosfolipídeos/químicaRESUMO
The structure and thermal stability of a laccase from Rigidoporus lignosus (Rl) was analysed by Fourier-transform infrared (FT-IR) spectroscopy. The enzyme was depleted of copper atoms, then part of the apoenzyme was re-metalled and these two forms of the protein were analysed as well. The enzymatic activity, lost by the removal of copper atoms, was restored in the re-metalled apoenzyme and resulted similar to that of native protein. The infrared data indicated that the enzyme contains a large amount of beta-sheets and a small content of alpha-helices, and it displayed a marked thermostability showing the T(m) at 92.5 degrees C. The apoenzyme and the re-metalled apoenzyme did not show remarkable differences in the secondary structure with respect to the native protein, but the thermal stability of the apoenzyme was dramatically reduced showing a T(m) close to 72 degrees C, while the re-metalled protein displayed the T(m) at 90 degrees C. These data indicate that copper atoms, beside their role in catalytic activity, play also an important role on the stabilisation of the structure of Rl laccase. About 35% of the polypeptide chain is buried and/or constitutes a particular compact structure, which, beside copper atoms, is probably involved in the high thermal stability of the protein. Another small part of the structure is particularly sensitive to high temperatures and it could be the cause of the loss of enzymatic activity when the temperature is raised above 45-50 degrees C.
Assuntos
Oxirredutases/química , Oxirredutases/metabolismo , Polyporales/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Temperatura Alta , Cinética , Lacase , Desnaturação Proteica , Espectrofotometria , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , TermodinâmicaRESUMO
The structural properties and the conformational dynamics of antarctic fish Notothenia coriiceps and mouse metallothioneins were studied by Fourier-transform infrared and fluorescence spectroscopy. Infrared data revealed that the secondary structure of the two metallothioneins is similar to that of other metallothioneins, most of which lack periodical secondary structure elements such as alpha-helices and beta-sheets. However, the infrared spectra of the N. coriiceps metallothionein indicated the presence of a band, which for its typical position in the spectrum and for its sensitivity to temperature was assigned to alpha-helices whose content resulted in 5% of the total secondary structure of the protein. The short alpha-helix found in N. coriiceps metallothionein showed an onset of denaturation at 30 degrees C and a T(m) at 48 degrees C. The data suggest that in N. coriiceps metallothionein a particular cysteine is involved in the alpha-helix and in the metal-thiolate complex. Moreover, infrared spectra revealed that both proteins investigated possess a structure largely accessible to the solvent. The time-resolved fluorescence data show that N. coriiceps metallothionein possesses a more flexible structure than mouse metallothionein. The spectroscopic data are discussed in terms of the biological function of the metallothioneins.