RESUMEN
Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang-actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo ("wild-type", wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein-copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1-17) and AcAng(1-17), which encompass the sequence 1-17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.
Asunto(s)
Complejos de Coordinación/farmacología , Cobre/metabolismo , Neuroblastoma/patología , Fragmentos de Péptidos/farmacología , Ribonucleasa Pancreática/metabolismo , Sitios de Unión , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Concentración de Iones de Hidrógeno , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Unión Proteica , Ribonucleasa Pancreática/química , Espectrofotometría UltravioletaRESUMEN
In this work we address the question whether light-induced changes in the Mg(II) content in the chloroplast lumen can modulate the electron donation to photosystem I, in particular the electrostatic interaction between plastocyanin (Pc) and the photosystem 1 subunit PsaF. For this, we have used 2D NMR spectroscopy to study the binding of Mg(II) ions and the isolated luminal domain of PsaF to (15)N-labelled Pc. From the chemical-shift perturbations in the (1)H-(15)N HSQC spectra, dissociation constants of (4.9 ± 1.7) mM and (1.4 ± 0.2) mM were determined for the Pc-Mg(II) and Pc-PsaF complexes, respectively. In both cases, significant chemical-shift changes were observed for Pc backbone amide groups belonging to the two acidic patches, residues 42-45 and 59-61. In addition, competitive effects were observed upon the addition of Mg(II) ions to the Pc-PsaF complex, further strengthening that Mg(II) and PsaF bind to the same region on Pc. To structurally elucidate the Mg(II) binding site we have utilized Mn(II) as a paramagnetic analogue of Mg(II). The paramagnetic relaxation enhancement induced by Mn(II) results in line broadening in the Pc HSQC spectra which can be used to estimate distances between the bound ion and the affected nuclear spins. The calculations suggest a location of the bound Mn(II) ion close to Glu43 in the lower acidic patch, and most likely in the form of a hexaquo complex embedded within the hydration shell of Pc. The results presented here suggest a specific binding site for Mg(II) that may regulate the binding of Pc to photosystem 1 in vivo.
Asunto(s)
Magnesio/metabolismo , Manganeso/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/metabolismo , Plastocianina/metabolismo , Spinacia oleracea/metabolismo , Cloroplastos/química , Cloroplastos/metabolismo , Luz , Magnesio/química , Manganeso/química , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plastocianina/química , Plastocianina/genética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The angiogenin protein (hAng) is a potent angiogenic factor and its cellular activities may be affected by copper ions even if it is yet unknown how this metal ion is able to produce this effect. Among the different regions of hAng potentially able to bind copper ions, the N-terminal domain appears to be an ideal candidate. Copper(II) complexes of the peptide fragments encompassing the amino acid residues 4-17 of hAng protein were characterized by potentiometric, UV-vis, CD, and EPR spectroscopic methods. The results show that these fragments have an unusual copper(II) binding ability. At physiological pH, the prevailing complex species formed by the peptide encompassing the protein sequence 4-17 is [CuHL], in which the metal ion is bound to two imidazole and two deprotonated amide nitrogen atoms disposed in a planar equatorial arrangement. Preliminary spectroscopic (UV-vis, CD, and EPR) data obtained on the copper(II) complexes formed by the whole recombinant hAng protein, show a great similarity with those obtained for the N-terminal peptide fragments. These findings indicate that within the N-terminal domain there is one of the preferred copper(II) ions anchoring site of the whole recombinant hAng protein.
Asunto(s)
Cobre/metabolismo , Fragmentos de Péptidos/metabolismo , Ribonucleasa Pancreática/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Fragmentos de Péptidos/química , Potenciometría , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleasa Pancreática/química , Espectrofotometría UltravioletaRESUMEN
Angiogenin (Ang) is a potent angiogenic protein that is overexpressed in many types of cancer at concentration values correlated to the tumor aggressiveness. Here, by means of an integrated multi-technique approach based on crystallographic, spectrometric and spectroscopic analyses, we demonstrate that the anti-cancer drug oxaliplatin efficiently binds angiogenin. Microscopy cellular studies, carried out on the prostate cancer cell (PC-3) line , show that oxaliplatin inhibits the angiogenin prompting effect on cell proliferation and migration, which are typical features of angiogenesis process. Overall, our findings point to angiogenin as a possible target of oxaliplatin, thus suggesting a potential novel mechanism for the antineoplastic activity of this platinum drug and opening the avenue to novel approaches in the combined anti-cancer anti-angiogenic therapy.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Oxaliplatino/farmacología , Neoplasias de la Próstata , Ribonucleasa Pancreática/metabolismo , Humanos , Masculino , Células PC-3 , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismoRESUMEN
The new Ru complex 8 containing the bio-inspired ligand 7 was successfully synthesized and characterized. Complex 8 efficiently catalyzes water oxidation using Ce(IV) and Ru(III) as chemical oxidants. More importantly, this complex has a sufficiently low overpotential to utilize ruthenium polypyridyl-type complexes as photosensitizers.
Asunto(s)
Ligandos , Luz , Rutenio/química , Agua/química , Cerio/química , Estructura Molecular , Oxidación-Reducción , Oxígeno/química , FotoquímicaRESUMEN
The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and ¹5N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.
Asunto(s)
Complejo de Proteína del Fotosistema I/química , Proteínas de Plantas/química , Proteínas Recombinantes de Fusión/química , Spinacia oleracea/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Clonación Molecular , Citocromos c6/química , Citocromos c6/metabolismo , Disulfuros , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Concentración Osmolar , Fragmentos de Péptidos , Complejo de Proteína del Fotosistema I/biosíntesis , Complejo de Proteína del Fotosistema I/aislamiento & purificación , Complejo de Proteína del Fotosistema I/metabolismo , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Plastocianina/química , Plastocianina/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Spinacia oleracea/química , Espectrometría de Masas en Tándem , Temperatura , TiorredoxinasRESUMEN
In this work, we aimed to develop a hybrid theranostic nano-formulation based on gold nanoparticles (AuNP)-having a known anti-angiogenic character-and the angiogenin (ANG), in order to tune the angiogenesis-related phases involved in the multifaceted process of the wound healing. To this purpose, spherical were surface "decorated" with three variants of the protein, namely, the recombinant (rANG), the wild-type, physiologically present in the human plasma (wtANG) and a new mutant with a cysteine substitution of the serine at the residue 28 (S28CANG). The hybrid biointerface between AuNP and ANG was scrutinized by a multi-technique approach based on dynamic light scattering, spectroscopic (UV-visible, circular dichroism) and microscopic (atomic force and laser scanning confocal) techniques. The analyses of optical features of plasmonic gold nanoparticles allowed for discrimination of different adsorption modes-i.e.; predominant physisorption and/or chemisorption-triggered by the ANG primary sequence. Biophysical experiments with supported lipid bilayers (SLB), an artificial model of cell membrane, were performed by means of quartz crystal microbalance with dissipation monitoring acoustic sensing technique. Cellular experiments on human umbilical vein endothelial cells (HUVEC), in the absence or presence of copper-another co-player of angiogenesis-were carried out to assay the nanotoxicity of the hybrid protein-gold nanoassemblies as well as their effect on cell migration and tubulogenesis. Results pointed to the promising potential of these nanoplatforms, especially the new hybrid Au-S28CANG obtained with the covalent grafting of the mutant on the gold surface, for the modulation of angiogenesis processes in wound care.
RESUMEN
The doppel protein (Dpl) is the first homologue of the prion protein (PrP(C)) to be discovered; it is overexpressed in transgenic mice that lack the prion gene, resulting in neurotoxicity. The whole prion protein is able to inhibit Dpl neurotoxicity, and its N-terminal domain is the determinant part of the protein function. This region represents the main copper(II) binding site of PrP(C). Dpl is able to bind at least one copper ion, and the specific metal-binding site has been identified as the histidine residue at the beginning of the third helical region. However, a reliable characterization of copper(II) coordination features has not been reported. In a previous paper, we studied the copper(II) interaction with a peptide that encompasses only the loop region potentially involved in metal binding. Nevertheless, we did not find a complete match between the EPR spectroscopic parameters of the copper(II) complexes formed with the synthesized peptide and those reported for the copper(II) binding sites of the whole protein. Herein, the synthesis of the human Dpl peptide fragment hDpl(122-139) (Ac-KPDNKLHQQVLWRLVQEL-NH(2)) and its copper(II) complex species are reported. This peptide encompasses the third alpha helix and part of the loop linking the second and the third helix of human doppel protein. The single-point-mutated peptide, hDpl(122-139)D124N, in which aspartate 124 replaces an asparagine residue, was also synthesized. This peptide was used to highlight the role of the carboxylate group on both the conformation preference of the Dpl fragment and its copper(II) coordination features. NMR spectroscopic measurements show that the hDpl(122-139) peptide fragment is in the prevailing alpha-helix conformation. It is localized within the 127-137 amino acid residue region that represents a reliable conformational mimic of the related protein domain. A comparison with the single-point-mutated hDpl(122-139)D124N reveals the significant role played by the aspartic residue in addressing the peptide conformation towards a helical structure. It is further confirmed by CD measurements. Potentiometric titrations were carried out in aqueous solutions to obtain the stability constant values of the species formed by copper(II) with the hDpl peptides. Spectroscopic studies (EPR, NMR, CD, UV/Vis) were performed to characterize the coordination environments of the different metal complexes. The EPR parameters of the copper(II) complexes with hDpl(122-139) match those of the previously reported copper(II) binding sites of the whole hDpl. Addition of the copper(II) ion to the peptide fragment does not alter the helical conformation of hDpl(122-139), as shown by CD spectra in the far-UV region. The aspartate-driven preorganized secondary structure is not significantly modified by the involvement of Asp124 in the copper(II) complex species that form in the physiological pH range. To elaborate on the potential role of copper(II) in the recently reported interaction between the PrP(C) and Dpl, the affinity of the copper(II) complexes towards the prion N terminus domain and the binding site of Dpl was reported.
Asunto(s)
Cobre/química , Priones/química , Secuencia de Aminoácidos , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Ligadas a GPI/química , Humanos , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Espectrofotometría Ultravioleta , TermodinámicaRESUMEN
In bright light the photosynthetic reaction center (RC) of Rhodobacter sphaeroides stabilizes the P(+)(870).Q(-)(A) charge-separated state and thereby minimizes the potentially harmful effects of light saturation. Using X-ray diffraction we report a conformational change that occurs within the cytoplasmic domain of this RC in response to prolonged illumination with bright light. Our observations suggest a novel structural mechanism for the regulation of electron transfer reactions in photosynthesis.
Asunto(s)
Luz , Modelos Moleculares , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Rhodobacter sphaeroides/metabolismo , Cristalografía , Transporte de Electrón/fisiología , Fotoquímica , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Conformación Proteica , Difracción de Rayos XRESUMEN
The electron transfer from wild-type spinach plastocyanin (Pc) to photosystem 1 has been studied by flash-induced absorption changes at 830 nm. The decay kinetics of photo-oxidized P700 are drastically slower in the presence of Ag(I)-substituted Pc, while addition of Zn(II)-substituted Pc has a weaker effect. The metal-substituted forms of Pc act as competitive inhibitors of the reaction between normal, Cu-containing, Pc and P700. The inhibition constants obtained from an analysis of the kinetic data were 30 and 410 microM for Ag(I)- and Zn(II)-substituted Pc, respectively. When the Gly8Asp mutant form of Pc was used instead of the wild-type form, the corresponding values were found to be 77 and 442 microM. If the Ag- and Zn-derivatives can be considered as structural mimics of reduced and oxidized CuPc, respectively, our results imply that there is a redox-induced decrease in the affinity between Pc and photosystem 1 that follows the electron donation to P700. Our data also imply that the Gly8Asp mutation can diminish the magnitude of this change. The findings reported here are consistent with a reaction mechanism where the electron transfer in the complex between Pc and photosystem 1 is assumed to be reversible.
Asunto(s)
Electrones , Metales/metabolismo , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/metabolismo , Spinacia oleracea/metabolismo , Cobre , Concentración de Iones de Hidrógeno , Cinética , Proteínas Mutantes/química , Oxidación-Reducción , Fotólisis , Plastocianina/química , Plastocianina/aislamiento & purificación , Estructura Secundaria de Proteína , Plata , Análisis Espectral , ZincRESUMEN
Plastocyanin (Pc) is a copper-containing protein, which functions as an electron carrier between the cytochrome b(6)f and photosystem 1 (PS1) complexes in the photosynthetic electron transfer (ET) chain. The ET is mediated by His87 situated in the hydrophobic surface in the north region of Pc. Also situated in this region is Leu12, which mutated to other amino acids severely disturbs the ET from cytochrome f and to PS1, indicating the importance of the hydrophobic surface. The crystal structure of the Pc double mutant G8D/L12E has been determined to 2.0 A resolution, with a crystallographic R-factor of 18.3% (R(free)=23.2%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. In particular, there is a small but significant change in the hydrophobic surface close to His87. Evidently, this leads to a mismatch in the reactive complex with the redox partners. For PS1 this results in a 20 times weaker binding and an eightfold slower ET as determined by kinetic measurements. The mutations that have been introduced do not affect the optical absorption spectrum. However, there is a small change in the EPR spectrum, which can be related to changes in the copper coordination geometry.
Asunto(s)
Plastocianina/química , Spinacia oleracea/química , Sustitución de Aminoácidos , Cristalografía por Rayos X , Citocromos f/metabolismo , Focalización Isoeléctrica , Cinética , Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/genética , Plastocianina/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Spinacia oleracea/genética , Spinacia oleracea/metabolismoRESUMEN
Angiogenin (ANG), a member of the secreted ribonuclease family, is a potent angiogenesis stimulator that interacts with endothelial cells inducing a wide range of responses. Metal ions dyshomeostasis play a fundamental role in the onset of neurodegenerative diseases, in particular copper that is also involved in angiogenesis processes. It is known that vascular pathologies are present in neurodegenerative diseases and Angiogenin is down-regulated in Alzheimer and Parkinson diseases, as well as it has been found as one of the mutated genes in amyotrophic lateral sclerosis (ALS). Copper (II) induces an increase of Angiogenin binding to endothelial cells but, so far, the relationship between copper-ANG and angiogenesis induction remain unclear. Herein, the effects of copper (II) ions on Angiogenin activity and expression were evaluated. The binding of copper was demonstrated to affect the intracellular localization of the protein decreasing its nuclear translocation. Moreover, the ANG-copper (II) system negatively affects the protein-induced angiogenesis, as well as endothelial cells migration. Surprisingly, copper also reveals the ability to modulate the Angiogenin transcription. These results highlight the tight relationship between copper and Angiogenin, pointing out the biological relevance of ANG-copper system in the regulation of endothelial cell function, and revealing a possible new mechanism at the basis of vascular pathologies.
Asunto(s)
Cobre/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Ribonucleasa Pancreática/metabolismo , Humanos , ARN Ribosómico/metabolismo , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/farmacologíaRESUMEN
In the photosynthetic electron-transfer chain, the photosystem I subunit PsaF is involved in the specific binding of plastocyanin. Using fluorescence electrophoresis we show here that the luminal domain of PsaF is a target for thioredoxin-mediated reduction of the Cys residues 8 and 63. Furthermore, by using NMR spectroscopy, we show that the thiolated form of PsaF has a lower affinity towards reduced plastocyanin than when the disulfide bridge is intact. Time-resolved absorbance measurements and fluorescence electrophoresis shows that oxidized plastocyanin can re-oxidize PsaF and thus restore the active form.
Asunto(s)
Complejo de Proteína del Fotosistema I/metabolismo , Plastocianina/metabolismo , Spinacia oleracea/enzimología , Tiorredoxinas/metabolismo , Cisteína/metabolismo , Disulfuros , Electroforesis , Oxidación-Reducción , Unión Proteica , Análisis EspectralRESUMEN
Angiogenin is one of the more potent angiogenic factors known, whose activity may be affected by the presence of copper ions. Copper(II) complexes with the peptides encompassing the putative endothelial cell binding domain of angiogenin, Ac-KNGNPHREN-NH(2) and Ac-PHREN-NH(2), have been characterized by potentiometric, UV-vis, CD and EPR spectroscopic methods. The coordination features of all the copper complex species derived by both peptides are practically the same, as predictable because of the presence of a proline residue within their aminoacidic sequence. In particular, Ac-PHREN-NH(2) is really the aminoacidic sequence involved in the binding to copper(II). Thermodynamic and spectroscopic evidence are given that side chain oxygen donor atom of glutamyl residue is involved in the copper binding up to physiological pH. EPR parameters suggest that the carboxylate group is still involved also in the predominant species [Cu(L)H(-2)], the metal coordination environment being probably formed by N(Im), 2N(-), H(2)O in equatorial plane and an oxygen atom from COO(-) in apical position, or vice versa, with the carboxylate oxygen atom in the copper coordination plane and the water molecule confined to one of the apical positions. Moreover, the comparison with the thermodynamic and spectroscopic results in the case of the copper(ii) complex species formed by the single point mutated peptide, Ac-PHRQN-NH(2), provides further evidence of the presence of carboxylate oxygen atom in the copper coordination sphere.
Asunto(s)
Cobre/química , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/metabolismo , Péptidos/química , Ribonucleasa Pancreática/metabolismo , Inductores de la Angiogénesis/química , Inductores de la Angiogénesis/metabolismo , Sitios de Unión , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Células Endoteliales/metabolismo , Concentración de Iones de Hidrógeno , Nitrógeno/química , Compuestos Organometálicos/química , Oxígeno/química , Potenciometría , Prolina/química , Ribonucleasa Pancreática/química , Espectrofotometría Ultravioleta , Termodinámica , Agua/químicaRESUMEN
Copper(II) complexes of the peptide fragment (Dpl122-130) encompassing the sequence 122-130 of human doppel protein were characterized by potentiometric, UV-Visible, CD and EPR spectroscopic methods. An analogous peptide, in which the aspartate residue was substituted by an asparagine amino acid, was synthesized in order to provide evidence on the possible role of carboxylate group in copper(II) coordination. It was found that the carboxylic group is directly involved in copper(II) coordination at acidic pH, forming the CuLH(2) species with Dpl122-130. This copper(II) complex displayed EPR parameters very similar to those of the analogous complex with the whole doppel protein. At pH higher than 7, the complexes showed magnetic parameters similar to those of the major species of protein formed in the pH range 7-8, with the metal coordination environment consisting of one imidazole and three amide nitrogen atoms. The comparison of Cu-Dpl122-130 binding constant values with those of the prion peptide fragments (PrP106-114), showed that doppel peptide had a higher metal binding affinity at acidic pH whereas the prion peptide fragment binds the metal tightly at physiological pH.
Asunto(s)
Cobre/química , Fragmentos de Péptidos/química , Priones/química , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Ligadas a GPI , Humanos , PotenciometríaRESUMEN
Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)(4)-NH(2), and the mutated residue, Ac-(PHNPGF)(4)-NH(2), to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)(4)-NH(2), was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat.
Asunto(s)
Cobre/química , Priones/química , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Pollos , Ratones , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Priones/genética , Análisis Espectral , TermodinámicaRESUMEN
Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.
Asunto(s)
Rodopsina , Reactores Biológicos , Clonación Molecular , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Expresión Génica , Resonancia Magnética Nuclear Biomolecular , Señales de Clasificación de Proteína , Bombas de Protones , Rodopsina/biosíntesis , Rodopsina/química , Rodopsina/genética , Rodopsina/aislamiento & purificación , Rodopsinas MicrobianasRESUMEN
Many properties of copper-containing nitrite reductase are pH-dependent, such as gene expression, enzyme activity, and substrate affinity. Here we use x-ray diffraction to investigate the structural basis for the pH dependence of activity and nitrite affinity by examining the type 2 copper site and its immediate surroundings in nitrite reductase from Rhodobacter sphaeroides 2.4.3. At active pH the geometry of the substrate-free oxidized type 2 copper site shows a near perfect tetrahedral geometry as defined by the positions of its ligands. At higher pH values the most favorable copper site geometry is altered toward a more distorted tetrahedral geometry whereby the solvent ligand adopts a position opposite to that of the His-131 ligand. This pH-dependent variation in type 2 copper site geometry is discussed in light of recent computational results. When co-crystallized with substrate, nitrite is seen to bind in a bidentate fashion with its two oxygen atoms ligating the type 2 copper, overlapping with the positions occupied by the solvent ligand in the high and low pH structures. Fourier transformation infrared spectroscopy is used to assign the pH dependence of the binding of nitrite to the active site, and EPR spectroscopy is used to characterize the pH dependence of the reduction potential of the type 2 copper site. Taken together, these spectroscopic and structural observations help to explain the pH dependence of nitrite reductase, highlighting the subtle relationship between copper site geometry, nitrite affinity, and enzyme activity.
Asunto(s)
Cobre/química , Nitrito Reductasas/química , Nitritos/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Nitrito Reductasas/metabolismo , Oxidación-Reducción , Unión Proteica , Conformación Proteica , Rhodobacter sphaeroides/enzimología , Espectroscopía Infrarroja por Transformada de Fourier , Especificidad por SustratoRESUMEN
Eukaryotic--especially human--membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP1 was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQP1 in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization.