Direct Observation of the Mechanical Role of Bacterial Chaperones in Protein Folding

Protein folding under force is an integral source of generating mechanical energy in various cellular processes, ranging from protein translation to degradation. Although chaperones are well known to interact with proteins under mechanical force, how they respond to force and control cellular energetics remains unknown. To address this question, we introduce a real-time magnetic tweezer technology herein to mimic the physiological force environment on client proteins, keeping the chaperones unperturbed. We studied two structurally distinct client proteins--protein L and talin with seven different chaperones-independently and in combination and proposed a novel mechanical activity of chaperones. We found that chaperones behave differently, while these client proteins are under force, than their previously known functions. For instance, tunnel-associated chaperones (DsbA and trigger factor), otherwise working as holdase without force, assist folding under force. This process generates an additional mechanical energy up to similar to 147 zJ to facilitate translation or translocation. However, well-known cytoplasmic foldase chaperones (PDI, thioredoxin, or DnaKJE) do not possess the mechanical folding ability under force. Notably, the transferring chaperones (DnaK, DnaJ, and SecB) act as holdase and slow down the folding process, both in the presence and absence of force, to prevent misfolding of the client proteins. This provides an emerging insight of mechanical roles of chaperones: they can generate or consume energy by shifting the energy landscape of the client proteins toward a folded or an unfolded state, suggesting an evolutionary mechanism to minimize energy consumption in various biological processes.