We have developed a suite of techniques for single-molecule force spectroscopy to observe the cleavage and reformation of disulfide and thioester bonds—one protein and one bond at a time. With atomic force microscope (AFM) or magnetic tweezers (MT)-based assays, we can monitor single bond cleavages in real time as discrete changes in the length of the unfolding or refolding parent protein. The mechanochemistry of disulfide and thioester bonds present two differing pictures of biological force-dependent reactivity. Cysteine-cysteine disulfide bonds are a ubiquitous structural feature in proteins and are commonly thought to serve a stabilizing function. We observe that various small molecules, including 1,4-DL-dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP), can reduce disulfide bonds with rates that increase with increasing force, offering insights into the mechanisms of each transition state. In contrast, certain oxidoreductase enzymes, such as human thioredoxin, reduce the disulfide bond with a negative force dependence, owing to the orientations of the stretched Cys-Cys bond within the enzyme active site. We have also developed an assay to investigate the mechanochemistry of thioester bonds that are ubiquitous in central metabolism. Studying a native cysteine-glutamine thioester bond in a bacterial adhesin, we find that thioester cleavage by small molecule amine nucleophiles is strongly inhibited by force, observable only at forces below ~25 pN. Notably, this low-force range is more typical for cellular processes, and may afford a physiological reactivity switch. Force spectroscopy provides for a novel and robust assay of the mechanochemistry of disulfide and thioester bonds that play central roles in the activity of numerous biological molecules and that are difficult to study using traditional bulk assays.

Single molecule force spectroscopy studies of disulfide and thioester bonds

254th National Meeting (2017)

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