RESUMEN
The misfolding and aggregation of a small, natively unfolded protein α-synuclein (α-syn) is presumably an important factor in the development of Parkinson's disease. However, the mechanism of α-syn aggregation into amyloid fibrils and their morphology are not well understood. To elucidate the aggregation kinetics and the morphology of aggregates by the use of fluorescent techniques the protein needs to be suitably labeled. In this study, using atomic force microscopy, we demonstrate a significant effect of fluorescent labels on the α-syn fibrillization process. We studied in detail the morphology of α-syn aggregates as a function of the composition of mixtures of labeled and wild type (WT) α-syn in solution using different types of fluorescent dyes. Although the overall charge of the fluorophores we used and their chemical structure varied significantly, the morphology of α-syn fibrils changed in a similar way in all cases. The increase in the fraction of labeled α-syn in solution led to shortening of the fibrils as compared to those from WT-only α-syn, whereas the height of the fibrils remained mainly unaffected. The twisted fibril morphology observed in the WT and A140C α-syn mutant completely disappeared when the A140C α-syn mutant was 100% fluorescently labeled.
Asunto(s)
Amiloide/metabolismo , Colorantes Fluorescentes/metabolismo , alfa-Sinucleína/metabolismo , Escherichia coli/metabolismo , Cinética , Microscopía de Fuerza Atómica/métodos , Enfermedad de Parkinson/metabolismo , Agregación Patológica de Proteínas/metabolismo , Coloración y Etiquetado/métodosRESUMEN
Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.
Asunto(s)
Nitrito Reductasas/química , Nitrito Reductasas/metabolismo , Alcaligenes/enzimología , Alcaligenes/genética , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cobre/química , Transferencia Resonante de Energía de Fluorescencia , Cinética , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Nitrito Reductasas/genética , Oxidación-Reducción , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolucionesRESUMEN
Generation of functionally active biomolecular monolayers is important in both analytical science and biophysical analyses. Our ability to monitor the redox-active state of immobilized proteins or enzymes at a molecular level, from which stochastic and surface-induced variations would be apparent, is impeded by comparatively slow electron-transfer kinetics and associated signal:noise difficulties. We demonstrate herein that by covalently tethering an appropriate dye to the copper protein azurin a highly oxidation-state-sensitive FRET process can be established which enables redox switching to be optically monitored at protein levels down to the zeptomolar limit. The surface-potential-induced cycling of emission enables the redox potential of clusters of a few hundred molecules to be determined.
Asunto(s)
Azurina/química , Biofisica/métodos , Química Física/métodos , Cobre/química , Electroquímica/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes/farmacología , Electrodos , Electrones , Cinética , Modelos Químicos , Conformación Molecular , Oxidación-Reducción , Estructura Terciaria de Proteína , Propiedades de SuperficieRESUMEN
We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.
Asunto(s)
Azurina/química , Azurina/ultraestructura , Solventes/química , Agua/química , Adsorción , Transporte de Electrón , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The assessment of the folding and of the structural stability of a protein in air, upon immobilization in the solid state, represents a critical point from both a fundamental point of view and for the development of solid state nanobioelectronics. The recent demonstrations by Rinaldi et al. [R. Rinaldi et al., Adv. Mater. 14, 1453 (2002); Appl. Phys. Lett. 82, 472 (2003); Ann. (N.Y.) Acad. Sci. 1006, 187 (2003)] of protein-based solid state devices and transistors working in air have raised an intriguing question about the behavior of a biomolecule under nonphysiological conditions. The operation principle of the realized devices is based on the physiological electron transfer function of the metalloprotein azurin. This means that azurin should retain its shape and functionality also in the solid state when utilized in air and at room temperature. In this Brief Report, we prove this claim by analyzing the conformational state of the azurin monolayers developed for such devices by means of intrinsic fluorescence spectroscopy. We show that the immobilization of azurins in the solid state under nonliquid conditions, by means of a specific chemisorption process, does not necessarily lead to protein denaturation. This result is of great importance because it opens up interesting perspectives for the development of solid state hybrid nanodevices for electronic applications requiring nonliquid environments.
Asunto(s)
Espectrometría de Fluorescencia/métodos , Adsorción , Aire , Estabilidad de Medicamentos , Unión Proteica , Conformación Proteica , Desnaturalización ProteicaRESUMEN
The study of the electronic conduction through plastocyanin (PC) mutants assembled on a gold surface has been addressed by scanning tunneling spectroscopy. The two mutants exploit a single thiol group (PCSH) or a disulfide bridge (PCSS) to covalently bind at gold surface. The I-V measurements were performed by positioning the STM tip on top of a single molecule and sweeping the bias potential between +/-1 V, under both ambient and controlled atmosphere. For PCSS, under ambient conditions, asymmetric I-V characteristics were obtained, which disappear under nitrogen atmosphere. PCSH, instead shows a symmetric I-V relation in air and under nitrogen environment. Here, as factors underlying this distinct electron conductive behaviour, a potential role for hydration water molecules and for copper redox levels are discussed.
Asunto(s)
Atmósfera/química , Oro/metabolismo , Microscopía de Túnel de Rastreo/métodos , Plastocianina/química , Aire , Cobre/química , Cobre/metabolismo , Disulfuros/química , Conductividad Eléctrica , Electroquímica , Transporte de Electrón , Mutación , Nitrógeno , Oxidación-Reducción , Plastocianina/genética , Plastocianina/metabolismo , Compuestos de Sulfhidrilo/química , Agua/química , Agua/metabolismoRESUMEN
An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.
Asunto(s)
Metaloproteínas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Azurina/química , Azurina/metabolismo , Proteínas Bacterianas/química , Cobre/química , Concentración de Iones de Hidrógeno , Metaloproteínas/metabolismo , Desnaturalización Proteica , Pliegue de Proteína , Termodinámica , Zinc/químicaRESUMEN
The effect of copper/zinc metal ion replacement on the folding free energy of wild type (w.t.) and disulfide bridge depleted (C3A/C26A) azurin has been investigated by differential scanning calorimetry (DSC) and fluorescence techniques. The denaturation experiments have shown that, in both cases, the thermal transitions of the zinc derivative of azurins can be depicted in terms of the classical Lumry-Eyring model, N if U-->F, thus resembling the unfolding path of the two copper proteins. The thermally induced transition of Zn azurin, monitored by fluorescence occurs at lower temperature than the DSC scans indicating that a local conformational rearrangement of the Trp microenvironment, takes place before protein denaturation. For Zn C3A/C26A azurin, the two techniques reveal the same transition temperature. Comparison of the thermodynamic data shows that the presence of Zn in the active site stabilises the three-dimensional structure of azurin only when the disulfide bridge is present. Compared to the copper form of the protein, the unfolding temperature of Zn azurin has increased by 4 degrees C, while the unfolding free energy, deltaG, is 31 kJ/mol higher. Both enthalpic and entropic factors contribute to the observed DeltaG increase. However, the copper/zinc replacement has no effect on the unfolding free energy of C3A/C26A azurin. Taking Cu azurin w.t. as the reference state, for both Cu and Zn C3A/C26A azurin the unfolding free energy is decreased by about 28 kJ/mol, indicating that metal substitution is not able to compensate the destabilising effect induced by the disulfide bridge depletion. It is noteworthy that the thermal denaturation of the Zn derivative, which thermodynamically is the most stable form of azurin, is also characterized by the highest value of the activation energy, E(a), as derived from the kinetic stability analysis.
Asunto(s)
Azurina/química , Cobre/química , Zinc/química , Alanina/química , Rastreo Diferencial de Calorimetría , Cisteína/química , Cinética , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Espectrometría de Fluorescencia , Temperatura , TermodinámicaRESUMEN
The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature. The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric <--> g and <--> A tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins. Such a reduction, quantified by the standard deviations sigma(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution. In particular, the comparison of the sigma(g axially) and sigma(A axially) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is. The thiocyanate effect is stronger than the azide one. Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein. The effect observed upon N(-)(3) and SCN(-) addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions.
Asunto(s)
Aniones , Azurina/química , Proteínas Bacterianas/química , Conformación Proteica , Sustitución de Aminoácidos , Azurina/genética , Azurina/metabolismo , Proteínas Bacterianas/metabolismo , Clonación Molecular , Simulación por Computador , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Metaloproteínas/química , Metaloproteínas/metabolismo , Mutagénesis Sitio-Dirigida , Distribución Normal , Paracoccus/genética , Paracoccus/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The review deals with recent advances in magnetic resonance spectroscopy (hf EPR and NMR) of paramagnetic metal centers in biological macromolecules. In the first half of our chapter, we present an overview of recent technical developments in the NMR of paramagnetic bio-macromolecules. These are illustrated by a variety of examples deriving mainly from the spectroscopy of metalloproteins and their complexes. The second half focuses on recent developments in high-frequency EPR spectroscopy and the application of the technique to copper, iron, and manganese proteins. Special attention is given to the work on single crystals of copper proteins.
Asunto(s)
Espectroscopía de Resonancia por Spin del Electrón/métodos , Espectroscopía de Resonancia Magnética/métodos , Sitios de Unión , Biofisica/métodos , Cobre/metabolismo , Modelos Moleculares , Modelos TeóricosRESUMEN
Aiming to achieve stable immobilization for a redox-active cupredoxin protein onto a gold substrate and its consequent molecular level monitoring by Scanning Tunnelling Microscopy (STM), we introduced a disulphide bridge within poplar plastocyanin, while avoiding the perturbation of its active site. We selected and modified residues Ile-21 to Cys and Glu-25 to Cys by structurally conservative mutagenesis. Optical absorption spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), and resonance raman scattering (RRS) results indicate that the active site of the Ile21Cys, Glu25Cys plastocyanin (PCSS) to a large extent retains the spectroscopic properties of the wild-type protein. Furthermore, the redox midpoint potential of the couple CuII/CuI in PCSS, determined by cyclic voltammetry was found to be +348 mV close to the wild-type value. The STM images display self-assembled PCSS molecules immobilised onto gold substrate. Moreover, the full potentiostatic control of the electron transfer reaction during STM imaging, suggests that the adsorbed molecule maintains essentially its native redox properties.
Asunto(s)
Oro/química , Plastocianina/química , Plastocianina/genética , Adsorción , Sitios de Unión , Cisteína/genética , Disulfuros/química , Electroquímica , Electrodos , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Microscopía de Túnel de Rastreo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Plastocianina/fisiología , Espectrofotometría , Espectrometría Raman , ÁrbolesRESUMEN
The complex of the photosynthetic redox partners plastocyanin and cytochrome f from the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and, to a lesser extent, the "eastern face" of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f. Rigid-body structure calculations using NMR-derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex are independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH results in limited reorganization of the complex interface, while the binding affinity is unaffected. Therefore, protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron-transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.
Asunto(s)
Cianobacterias/química , Citocromos/química , Plastocianina/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Citocromos/metabolismo , Citocromos f , Concentración de Iones de Hidrógeno , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Concentración Osmolar , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Plastocianina/metabolismo , Conformación Proteica , Propiedades de SuperficieRESUMEN
An electrospray ionisation (ESI) mass spectrometric method for the determination of the free energy (DeltaG) of unfolding of proteins is described. The method was tested using three blue copper proteins: wild type azurin, Cys-3Ala/Cys-26Ala (C3A/C26A) azurin mutant and wild-type amicyanin. The time course of the denaturation process of the proteins dissolved in methanol/water (50:50, v/v, pH 3.5) was followed by recording ESI mass spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the free energy for the unfolding process for the three proteins could be determined. To evaluate the reliability of the thermodynamic data obtained by the ESI mass spectrometric approach, the denaturation process was followed by UV-VIS spectroscopy. The two sets of data obtained by these independent methods were in good agreement indicating that the ESI-MS approach can be used to obtain reliable quantitative information about the protein unfolding process. In principle, this approach can be applied to other proteins and requires very low amounts of sample, due to the intrinsic sensitivity of mass spectrometry. This may prove particularly useful when the amount of sample available prevents the use of current methods.
Asunto(s)
Proteínas Bacterianas/química , Sustitución de Aminoácidos , Azurina/química , Cobre/análisis , Metaloproteínas/química , Desnaturalización Proteica , Proteínas Recombinantes/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrofotometría , TermodinámicaRESUMEN
Next to their natural electron transport capacities, c-type cytochromes possess low peroxidase and cytochrome P-450 activities in the presence of hydrogen peroxide. These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics. This study reports on the peroxidase activity of cytochrome c-550 from Paracoccus versutus and the loss of this activity in presence of H2O2. The rate-determining step in the peroxidase reaction of cytochrome c-550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron. The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H2O2, and the lack of a base catalyst such as the distal His of the peroxidases. This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 M H2O2. Steady-state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build-up of amino-acid radicals on the protein. The inactivation kinetics in the absence of reducing substrate are mono-exponential and shown to be concurrent with haem degradation up to 25 mM H2O2 (pH 8.0). At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H2O2, involving the formation of superoxide and ferrocytochrome c-550.
Asunto(s)
Grupo Citocromo c/metabolismo , Paracoccus/enzimología , Peroxidasa/metabolismo , Aminoácidos/química , Catálisis , Relación Dosis-Respuesta a Droga , Activación Enzimática , Guayacol/química , Guayacol/farmacología , Hemo/química , Histidina/química , Peroxidasa de Rábano Silvestre/metabolismo , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Cinética , Oxidación-Reducción , Unión Proteica , Superóxido Dismutasa/farmacología , Superóxidos/química , Factores de TiempoRESUMEN
In order to investigate the relationship between the rate of protein-protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross-linking was engineered by replacing the surface-exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide-linked homo-dimers of azurin. Based on NMR line-broadening experiments, the electron self-exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7) x 10(5)M(-1)s(-1), which is a seven-fold decrease relative to wild-type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within a dimer and intermolecular electron transfer between two dimers, the e.s.e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag-Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecular electron transfer reaction in these latter dimers can be eliminated. The e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determined. This rate is compatible with a Cu-Cu distance of 18 A or more, which is larger than the Cu - Cu distance of 15 A observed in the wild-type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu distance should be in the range of 17 A < rCu-Cu < 28 A, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross-linking may disturb the formation of such an optimal electron transfer complex.
Asunto(s)
Azurina/química , Azurina/genética , Proteínas Bacterianas/química , Algoritmos , Asparagina/genética , Secuencia de Bases , Cobre/química , Cisteína/genética , Dimerización , Transporte de Electrón , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Histidina/genética , Espectroscopía de Resonancia Magnética , Conformación Molecular , Mutagénesis , Plásmidos/genética , Pseudomonas aeruginosa/genética , Zinc/químicaRESUMEN
The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.
Asunto(s)
Azurina/química , Azurina/genética , Mutación Puntual , Termodinámica , Equilibrio Ácido-Base/genética , Alcaligenes , Sustitución de Aminoácidos/genética , Azurina/metabolismo , Sitios de Unión/genética , Cobre/metabolismo , Entropía , Concentración de Iones de Hidrógeno , Ligandos , Metionina/genética , Metionina/metabolismo , Oxidación-Reducción , Potenciometría/métodos , Pseudomonas aeruginosa , SolventesRESUMEN
The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.
Asunto(s)
Grupo Citocromo c/química , Citocromo-c Peroxidasa/química , Citocromos c , Proteínas de Saccharomyces cerevisiae , Sitios de Unión , Grupo Citocromo c/metabolismo , Citocromo-c Peroxidasa/metabolismo , Compuestos Férricos/química , Compuestos Ferrosos/química , Isoenzimas/química , Isoenzimas/metabolismo , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Oxidación-Reducción , Protones , Saccharomyces cerevisiae/enzimología , TermodinámicaRESUMEN
A novel blue copper protein was constructed by replacing the C-terminal loop of amicyanin (Paracoccus versutus) by the homologous loop of rusticyanin. The C-terminal loop of both amicyanin and rusticyanin contains three (His, Cys, Met) of the four copper ligands. The amicyanin mutant exhibits all spectroscopic properties normally encountered for blue copper sites. The midpoint potential (369 mV) is the highest reported value for an amicyanin mutant. Cyclic voltammetry and NMR studies of the reduced form indicate that, in contrast to wild-type amicyanin and all amicyanin mutants described so far, the C-terminal histidine ligand does not protonate in the accessible pH range (pKa<4.5).
Asunto(s)
Proteínas Bacterianas/química , Histidina/química , Proteínas Bacterianas/genética , Sitios de Unión , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Mutagénesis , Oxidación-Reducción , Paracoccus/química , Fotosíntesis , Conformación Proteica , ProtonesRESUMEN
Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c" isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS-PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and (1)H-NMR spectroscopy. The success in expressing the mature cytochrome c in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.
Asunto(s)
Clonación Molecular/métodos , Grupo Citocromo c/genética , Escherichia coli/genética , Methylophilus methylotrophus/genética , Familia de Multigenes , Rastreo Diferencial de Calorimetría , Genes Bacterianos , Vectores Genéticos , Proteínas Recombinantes/genética , Espectrofotometría UltravioletaRESUMEN
The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the development. In S. lividans disruption of ramABR resulted in the inability to produce aerial hyphae. Conversely, the identical mutant of S. coelicolor retained its developmental capacities, indicating the presence of a ram-independent developmental route that is not present or not activated in S. lividans. Aerial hyphae and spore formation in the S. lividans ramABR mutant was restored when grown near wild-type strains, suggesting that the ram gene products are involved in transport of a factor essential for normal development. In addition, an elevated copper concentration in the medium also relieved the developmental block of these mutants. These findings suggest that higher copper concentrations render this ram-associated factor obsolete.