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1.
J Biol Chem ; 290(23): 14518-27, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25897077

RESUMO

Oxidative folding, the process by which proteins fold and acquire disulfide bonds concurrently, is of critical importance for a wide range of biological processes. Generally, this process is catalyzed by oxidoreductase enzymes that facilitate oxidation and also bear chaperone functionality. Although this process has been well described qualitatively, fine yet important details remain obscured by a limited quantitative perspective, arising from the limitations in the application of bulk biochemical methods to the study of oxidative folding. In this work, we have applied single molecule force spectroscopy techniques to monitor in real time the process of oxidative folding as catalyzed by DsbA, the enzyme solely responsible for the catalysis of oxidative folding in the bacterial periplasm. We provide a quantitative and detailed description of the catalytic mechanism utilized by DsbA that offers insight into the entire sequence of events that occurs in the periplasm from the unfolded-reduced state to the folded-oxidized protein. We have compared our results with those of protein disulfide-isomerase, the eukaryotic counterpart of DsbA, allowing us to devise a general mechanism for oxidative folding that also reflects upon the physiological functions and demands of these enzymes in vivo.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Isomerases de Dissulfetos de Proteínas/química , Dobramento de Proteína , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia de Força Atômica , Oxirredução , Isomerases de Dissulfetos de Proteínas/metabolismo
2.
J Biol Chem ; 289(39): 26722-26732, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25096579

RESUMO

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.


Assuntos
Esclerose Lateral Amiotrófica/enzimologia , Cisteína/química , Dissulfetos/química , Mutação de Sentido Incorreto , Desdobramento de Proteína , Superóxido Dismutase/química , Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/genética , Cisteína/genética , Humanos , Oxirredução , Estrutura Secundária de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase-1
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