CHAPTER 1.3. Real-time Detection of Thiol Chemistry in Single Proteins

Abstract

The cell is exquisitely tuned to alter its functions and gene expression in response to changes in its redox environment. Initiation of signaling pathways thus requires proteins that are sensitive to redox conditions, which is usually achieved through strategic placement of cysteine residues within the protein. A major challenge for protein biochemists studying these pathways is to detect changes in the redox status in one or several cysteine residues in a protein in a reliable and time-resolved manner. Many techniques used to detect the redox state of a particular amino acid require treatment of the protein samples with chemical probes that react with side-chains of a certain oxidation state. The probes do not always react with absolute specificity for the side-chains, and there is always concern that the redox state of the side-chains may change during the isolation and treatment process. Single-molecule force spectroscopy (SMFS) provides a means for executing ChApTer 1.3

Cutting-Edge Single-Molecule Technologies Unveil New Mechanics in Cellular Biochemistry

“…DsbA and PDI are common examples of such enzymes. Overall, these proteins, although they are functionally similar to thioredoxin, are approximately 30,000 times faster than DTT (21). While millimolar concentrations of DTT are required, micromolar concentrations of thioredoxin are sufficient to reduce the protein.…”

“…Since AFM uses force to unfold the substrate, it is possible to study the impact of various redox enzymes on these bonds without using chemicals or heat. Unfolding of titin Ig domain I27 (I27G32C-A75C) in the denaturation pulse leads to only partial unfolding of the substrate, which yields an extension of 11 nm (21,51,52). Forces higher than 1 nN are needed to completely unfold and cleave covalent bonds (like disulphide bonds) (31).…”

“…Enzymes known as oxidoreductases play important roles in intracellular disulfide shuffling by forming or reducing disulfide bonds. Although they are ubiquitously present among different species, they have a conserved -Cys-X-X-Cys-motif present in their active sites, known as thioredoxin domains (21). DsbA and PDI are common examples of such enzymes.…”