Direct observation of the mechanical role of bacterial chaperones in protein folding

Abstract

Protein folding under force is an integral source of generating mechanical power to carry out diverse cellular functions. Though chaperones interact with proteins throughout the different stages of folding pathways, how they behave and interact with client proteins under force was not known. Here we introduce the ‘mechanical role’ of chaperone and explained it with seven independent chaperones using single molecule based real-time microfluidics-magnetic-tweezers. We showed and quantified how chaperones increase or decrease mechanical work output by shifting the folding energy landscape of the client proteins towards the folded or unfolded state. Notably, we found chaperones could behave differently under force. For instance: trigger factor, ribosomal-tunnel associated chaperone, working as a holdase in absence of force, but assist folding under force. This phenomenon generates extra mechanical energy to pull the polyprotein from the stalled ribosome. This is also relevant for SecYEG tunnel associated oxidoreductase DsbA, which act similarly like TF and increases the mechanical energy up to ~59 zJ, to facilitate membrane translocation in an energy efficient manner. However cytoplasmic oxidoreductases such as PDI and Thioredoxin, unlike DsbA, do not have the mechanical folding ability. Interestingly, we observed a highly potential foldase- DnaKJE chaperone complex, only restores the folding ability of the client protein and fails to act like TF or DsbA under force. However, the individual components of this complex, DnaK or DnaJ, act as a mechanical holdase and inhibits folding; similar to that of SecB. Together our study provides an emerging insight of mechanical chaperone behavior, where tunnel associated chaperones generate extra mechanical work whereas the cytoplasmic chaperones are unable to generate that, which might have evolved to minimize the energy consumption in biological processes.

Real time observation of chaperone-modulated talin mechanics with single molecule resolution

“…Such as oxidoreductase DsbA, a well-known model mechanical foldase, has the ability to increase the folding rate and mechanical stability of R3. 24,37,38 Hence, our results demonstrate that chaperones could modulate the mechanical stability of the FA protein talin, which are characterized by the change in the free energy barrier. Additionally, we propose this change in mechanical stability of R3 domain, can significantly affect binding kinetics with RIAM and vinculin: foldase chaperones facilitate the RIAM binding by shifting the energy barrier towards folded state and slows the stepwise unfolding of the domain, while unfoldase chaperones promote vinculin binding by decreasing the unfolding free energy barrier.…”

“…For in vitro biotinylation of the polyprotein, Avidity biotinylation kit was used and the biotinylated polypeptide was purified by Superdex-200 increase 10/300 GL gel filtration column in presence of Na-P buffer with 150 mM NaCl. 38…”