Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining ...

The existence of the phenomenon of enhanced enzyme diffusion (EED) has been a topic of debate in recent literature. One proposed mechanism to explain the origin of EED is oligomeric enzyme dissociation. We used mass photometry (MP), a label-free single-molecule technique, to investigate the dependence of the oligomeric states of several enzymes on their ligands. The studied enzymes of interest are catalase, aldolase, alkaline phosphatase, and vanillyl-alcohol oxidase (VAO). We compared the ratios of oligomeric states in the presence and absence of the substrate as well as different substrate and inhibitor concentrations. Catalase and aldolase were found to dissociate into smaller oligomers in the presence of their substrates, independently of inhibition, while for alkaline phosphatase and VAO, different behaviors were observed. Thus, we have identified a possible mechanism which explains the previously observed diffusion enhancement in vitro. This enhancement may occur due to the dissociation of oligomers through ligand binding.

An exploration of the unanswered questions in how the molecular chaperone Hsp90 supports protein homeostasis, and how single-molecule techniques could drive future breakthroughs in answering them.