Direct observation of disulfide isomerization in a single protein

• Published in: Nature chemistry Vol. 3; no. 11; pp. 882 - 887
• Main Authors: Alegre-Cebollada, Jorge, Kosuri, Pallav, Rivas-Pardo, Jaime Andrés, Fernández, Julio M.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.10.2011; Nature Publishing Group
• Subjects: 639/638/45/56; 639/638/92/469; Analytical Chemistry; Biochemistry; Chemical bonds; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Cysteine - chemistry; Disulfides - chemistry; Inorganic Chemistry; Isomerism; Kinetics; Microscopy, Atomic Force; Organic Chemistry; Oxidation; Oxidation-Reduction; Photochemicals; Physical Chemistry; Polypeptides; Protein Unfolding; Proteins; Proteins - chemistry; Proteins - metabolism; Spectroscopy; Sulfhydryl Compounds - chemistry; Sulfur; New York; United States > US
• ISSN: 1755-4330; 1755-4349; 1755-4349
• DOI: 10.1038/nchem.1155

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. Multiple redox reaction pathways exist in proteins containing several cysteines. A technique termed mechanical uncaging is now demonstrated, allowing the release of a single reactive cysteine within a protein and the unequivocal observation of subsequent thiol/disulfide exchanges. Mechanical uncaging of reactive groups is useful for studying chemical kinetics in a synchronized manner.