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

Abstract

Recent single molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (less than 5pN), folded talin binds RIAM for integrin activation; whereas at high force (more than 5pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic large protein, talin must interact with various chaperones, however the role of chaperones on talin mechanics is unknown. To address this question, we investigated the force response of a mechanically stable talin domain, with a set of well-known holdase and foldase chaperones, using a single molecule magnetic tweezers technology. Our findings demonstrate a novel mechanical role of chaperones. We found holdase chaperones reduce the mechanical stability of the protein to ~6 pN, while the foldase chaperone increases it up to ~15 pN. The alteration in mechanical stability ascribes to the underlying molecular mechanism where the chaperones directly reshape the energy landscape of talin. For example, unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.69 kBT and increases the refolding barrier from 3.49 to 11.31 kBT. In contrast, foldase chaperone (DsbA) increases the unfolding barrier to 33.46 kBT and decreases the refolding barrier to 0.44 kBT. The quantitative mapping of the chaperone-induced free energy landscape of talin directly shows that chaperones could perturb the focal adhesion dynamics, which in turn can influence downstream signaling cascades in diverse cellular processes.

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

“…The details of force calibration can be found in the supplemental information (Supplementary Figure 10-12). 22,27,28 In short, the force magnitude was calibrated by changing the magnetbead distance such as moving down the magnet position from 4.0 mm to 1.4 mm results in an increase in force pulse from 4.3 pN to 45 pN (Fig. 1A).…”