Direct observation of disulfide isomerization in a single protein.
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.
Department of Biological Sciences, Columbia University, Northwest Corner Building, 550 West 120 Street, New York, New York                 10027, USA.
Jorge
J
Alegre-Cebollada
Pallav
P
Kosuri
Jaime Andrés
JA
Rivas-Pardo
Julio M
JM
Fernández
D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D007536:Isomerism; D007700:Kinetics; D018625:Microscopy, Atomic Force; D010084:Oxidation-Reduction; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D011506:Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine