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

Abstract

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 ∼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.

Force-regulated chaperone activity of BiP/ERdj3 is opposite to their homologs DnaK/DnaJ: explained by strain energy

“…Similarly, after transforming into BL21 (DE3) cells, DnaK and DnaJ were grown in LB medium with respective antibiotics, centrifuged into pellet and resuspended in Na-phosphate buffer. Thereafter, the chaperones are purified according to the protocol described previously (17, 41).…”

“…Magnetic tweezers experiments were carried out in a fluid chamber where two different size of coverslips were sandwiched using parafilm strips as the spacer. The glass coverslips were cleaned exactly as described previously (41). In short, the glass slides were washed with 1.5% Hellmanex III solution and washed with double-distilled water.…”

“…The chambers were then treated with a blocking buffer (20 mM Tris-HCl of pH 7.4, 150 mM NaCl, 2 mM MgCl 2 , 1% BSA, and 0.03% NaN 3 ) to avoid non-specific interaction and kept in room temperature for 5 hours. These reactive fluid chambers were used for further experimentations by mounting them on a stage, controlled by a nanofocusing piezo actuator (P-725; Physik Instrumente), of an inverted microscope with oil immersion objective (9, 16, 41). Above the fluid chamber permanent neodymium magnets are attached to a voice coil actuator which regulate the distance of the magnets finally controlling the applied force.…”

“…19). The folded conformation of protein L (1HZ6) was chosen and unfolded conformation of protein L substrate was prepared using steered molecular dynamics simulation in NAMD, as described previously (41). Only AlphaFold structures of ERdj3 and DnaJ were chosen due to unavailability of their crystallographic structures.…”

Methotrexate-modulated talin-dynamics drives cellular mechanical phenotypes via YAP signaling

“…Owing to this magnetic manipulation, the force can be specifically applied on the substrate (Fig. 1A) and has been calibrated using the magnet law which has been extensively described in the literature [14][15][16][17][18][19] . While applying a force-ramp scan on the talin domain with a loading rate of 3.1 pN/s, we observed their unfolding event as a rapid extension at a certain force, which can be estimated from the force-extension curve.…”