期刊
NATURE CHEMISTRY
卷 3, 期 11, 页码 882-887出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEM.1155
关键词
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资金
- National Institutes of Health [HL66030, HL61228]
- Fundacion Caja Madrid
- Fundacion Alfonso Martin Escudero (Madrid, Spain)
- Fundacion Ibercaja (Zaragoza, Spain)
- la Comision Nacional de Investigacion Cientifica y Tecnologica
- Programa de Mejoramiento de la Calidad y la Equidad de la Educacion (Chile) [UCH7013]
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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