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1.
Anal Biochem ; 519: 57-70, 2017 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-27993553

RESUMEN

Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the ubiquitin to a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of protein turnover. Their specificities are only partially understood. We chemically synthesized ubiquitin, attached it to lysines belonging to the protein sequences known to be ubiquitinated. We chose the model protein "murine double minute 2" (mdm2), a ubiquitin ligase, itself ubiquitinated and deubiquitinated. We folded the ubiquitinated peptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which ubiquitin is attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.


Asunto(s)
Lisina/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteasas Ubiquitina-Específicas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Cromatografía en Gel , Dicroismo Circular , Humanos , Lisina/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , Proteolisis , Proteínas Proto-Oncogénicas c-mdm2/química , Especificidad por Sustrato , Ubiquitina/química , Ubiquitina-Proteína Ligasas/metabolismo
2.
Inorg Chem ; 53(15): 8071-82, 2014 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-25029381

RESUMEN

Cobaloximes are popular H2 evolution molecular catalysts but have so far mainly been studied in nonaqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties, {Co(dmgH)2} and {Co(dmgBF2)2} (dmgH2 = dimethylglyoxime), to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in the axial position of the cobalt complex, resulting in thermodynamically stable complexes. Quantum chemical/molecular mechanical docking calculations indicated a coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids, which provides them with the catalytic activity for H2 evolution in near-neutral aqueous conditions.


Asunto(s)
Hidrogenasas/química , Compuestos Organometálicos/química , Catálisis , Dicroismo Circular , Cobalto/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Simulación del Acoplamiento Molecular , Espectrofotometría Ultravioleta
3.
Chempluschem ; 81(10): 1083-1089, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31964078

RESUMEN

The insertion of cobaloxime catalysts in the heme-binding pocket of heme oxygenase (HO) yields artificial hydrogenases active for H2 evolution in neutral aqueous solutions. These novel biohybrids have been purified and characterized by using UV/visible and EPR spectroscopy. These analyses revealed the presence of two distinct binding conformations, thereby providing the cobaloxime with hydrophobic and hydrophilic environments, respectively. Quantum chemical/molecular mechanical docking calculations found open and closed conformations of the binding pocket owing to mobile amino acid residues. HO-based biohybrids incorporating a {Co(dmgH)2 } (dmgH2 =dimethylglyoxime) catalytic center displayed up to threefold increased turnover numbers with respect to the cobaloxime alone or to analogous sperm whale myoglobin adducts. This study thus provides a strong basis for further improvement of such biohybrids, using well-designed modifications of the second and outer coordination spheres, through site-directed mutagenesis of the host protein.

4.
Protein Sci ; 25(12): 2225-2242, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27670942

RESUMEN

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107-amino-acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X-ray diffraction. The refolded enzyme was compared to the recombinant, wild-type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N-terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure-function relationship of enzymes reachable by complete chemical synthesis.


Asunto(s)
Replegamiento Proteico , Proteínas de Unión a Tacrolimus , Cristalografía por Rayos X , Humanos , Dominios Proteicos , Relación Estructura-Actividad , Proteínas de Unión a Tacrolimus/síntesis química , Proteínas de Unión a Tacrolimus/química
5.
ChemSusChem ; 8(21): 3632-8, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26383700

RESUMEN

We describe here a systematic, reliable, and fast screening method that allows the comparison of H2-forming catalysts that work under aqueous conditions with two readily prepared chemical reductants and two commonly used photosensitizers. This method uses a Clark-type microsensor for H2 detection and complements previous methods based on rotating disk electrode measurements. The efficiencies of a series of H2 -producing catalysts based on Co, Ni, Fe, and Pt were investigated in aqueous solutions under thermal conditions with europium(II) reductants and under photochemical conditions in the presence of two different photosensitizers {[Ru(bipy)3]Cl2(bipy=2,2-bipyridine) and eosin-Y} and sacrificial electron donors (ascorbate and triethanolamine, respectively). The majority of catalysts tested were active only under specific conditions. However, our results also demonstrate the impressive versatility of a group of Co catalysts, which were able to produce H2 under different reducing conditions and at various pH values. In particular, a cobaloxime, [Co(dmgH)2(H2O)2] (dmgH2 =dimethylglyoxime), and a cobalt tetraazamacrocyclic complex, {Co(CR)Cl2}(+) [CR=2,12-dimethyl-3,7,11,17-tetraazabicylo(11.3.1)heptadeca-1(17),2,11,13,15-pentaene], displayed excellent catalytic rates under the studied conditions, and the best rates were observed under thermal conditions.


Asunto(s)
Hidrógeno/química , Metales Pesados/química , Compuestos Organometálicos/química , Agua/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Catálisis , Cobalto/química , Complejos de Coordinación , Europio/química , Hierro/química , Estructura Molecular , Níquel/química , Oxidación-Reducción , Fotoquímica , Platino (Metal)/química , Soluciones
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