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Direct observation of disulfide isomerization in a single protein

Abstract

Photochemical uncaging techniques use light to release active molecules from otherwise inert compounds. Here we expand this class of techniques by demonstrating the mechanical uncaging of a reactive species within a single protein. We proved this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond. We designed a protein that includes a caged cysteine and a buried disulfide. The mechanical unfolding of this protein in the presence of an external nucleophile frees the single reactive cysteine residue, which now can cleave the target disulfide via a nucleophilic attack on either one of its two sulfur atoms. This produces two different and competing reaction pathways. We used single-molecule force spectroscopy to monitor the cleavage of the disulfides, which extends the polypeptide by a magnitude unambiguously associated with each reaction pathway. This allowed us to measure, for the first time, the kinetics of disulfide-bond isomerization in a protein.

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Figure 1: Increased complexity in the reduction of a protein with two disulfides.
Figure 2: Fingerprints of the pathways available for the reduction of disulfide 24–55 after the reduction of disulfide 32–75.
Figure 3: Kinetic model of complete protein reduction by L-Cys following the initial reduction of disulfide 32–75.
Figure 4: The uncaging of a single cysteine residue in a protein allows calculation of the rate of spontaneous disulfide isomerization.

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Acknowledgements

This work was supported by National Institutes of Health grants HL66030 and HL61228 to J.M.F. J.A-C. thanks Fundación Caja Madrid, Fundación Alfonso Martín Escudero (Madrid, Spain) and Fundación Ibercaja (Zaragoza, Spain) for their financial support. J.A.R-P. is the recipient of a fellowship from la Comisión Nacional de Investigación Científica y Tecnológica and a Programa de Mejoramiento de la Calidad y la Equidad de la Educación Superior visiting scholar fellowship UCH7013 (Chile). We thank J. Li and B.J. Berne for their help with the steered molecular dynamics simulations. We also thank S.G-M. for reading the manuscript.

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J.A-C. and J.M.F. designed the research project, J.A-C. and J.A.R-P. performed the experiments, J.A-C., P.K., J.A.R-P. and J.M.F. analysed the data. J.A-C., P.K. and J.M.F. co-wrote the paper.

Corresponding authors

Correspondence to Jorge Alegre-Cebollada or Julio M. Fernández.

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The authors declare no competing financial interests.

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Alegre-Cebollada, J., Kosuri, P., Rivas-Pardo, J. et al. Direct observation of disulfide isomerization in a single protein. Nature Chem 3, 882–887 (2011). https://doi.org/10.1038/nchem.1155

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