title: Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA.
abstract: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biophysics, Columbia University Medical Center, New York, New York 10032.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Kahn
authors.affiliation: Department of Biological Sciences, Columbia University, New York, New York 10027.
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernández
authors.affiliation: Centro de Investigación Cooperativa (CIC) nanoGUNE, 20018 Donostia-San Sebastian, Spain, and IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain r.perezjimenez@nanogune.eu.
authors.forename: Raul
authors.initials: R
authors.lastname: Perez-Jimenez
mesh_terms: D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000378:metabolism; D017510:Protein Folding
chemical_list: D029968:Escherichia coli Proteins; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli
keywords: Escherichia coli (E. coli); atomic force microscopy (AFM); disulfide; enzyme kinetics; oxidation-reduction (redox); protein folding; single molecule biophysics