Cells continually sample their mechanical environment using exquisite force sensors such as talin, whose folding status triggers mechanotransduction pathways by recruiting binding partners. Mechanical signals in biology change quickly over time and are often embedded in noise; however, the mechanics of force-sensing proteins have only been tested using simple force protocols, such as constant or ramped forces. Here, using our magnetic tape head tweezers design, we measure the folding dynamics of single talin proteins in response to external mechanical noise and cyclic force perturbations. Our experiments demonstrate that talin filters out external mechanical noise but detects periodic force signals over a finely tuned frequency range. Hence, talin operates as a mechanical band-pass filter, able to read and interpret frequency-dependent mechanical information through its folding dynamics. We describe our observations in the context of stochastic resonance, which we propose as a mechanism by which mechanosensing proteins could respond accurately to force signals in the naturally noisy biological environment.

Vinculin binds unfolded talin domains in focal adhesions, which recruits actin filaments to reinforce the mechanical coupling of this organelle. However, it remains unknown how this interaction is regulated and its impact on the force transmission properties of this mechanotransduction pathway. Here, we use magnetic tweezers to measure the interaction between vinculin head and the talin R3 domain under physiological forces. For the first time, we resolve individual binding events as a short contraction of the unfolded talin polypeptide caused by the reformation of the vinculin-binding site helices, which dictates a biphasic mechanism that regulates this interaction. Force favors vinculin binding by unfolding talin and exposing the vinculin-binding sites; however, the coil-to-helix contraction introduces an energy penalty that increases with force, defining an optimal binding regime. This mechanism implies that the talin-vinculin-actin association could operate as a negative feedback mechanism to stabilize force on focal adhesions.

Uropathogenic Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent β-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains.

Gram-positive bacteria colonize mucosal tissues against large mechanical perturbations, such as coughing, which generate shear forces that exceed the ability of non-covalent bonds to remain attached. To overcome these challenges, the pathogen Streptococcus pyogenes utilizes the protein Cpa, a pilus tip-end adhesin equipped with a Cys-Gln thioester bond. The reactivity of this bond towards host surface ligands enables covalent anchoring; however, colonization also requires cell migration and spreading over surfaces. The molecular mechanisms underlying these seemingly incompatible requirements remain unknown. Here, we demonstrate a magnetic tweezers force spectroscopy assay that resolves the dynamics of Cpa thioester bond under force. While folded at forces < 6 pN, Cpa thioester bond reacts reversibly with amine ligands, that are a common occurrence in inflammation sites; however, mechanical unfolding and exposure to forces > 6 pN block thioester reformation. We hypothesize that this folding-coupled reactivity switch—“smart covalent bond”—could allow the adhesin to undergo binding and unbinding to surface ligands under low force and remain covalently attached under mechanical stress.

Cell-surface proteins are central for the interaction of cells with their surroundings and are also associated with numerous diseases. These molecules are exposed to mechanical forces, but the exact relation between force and the functions and pathologies associated with cell-surface proteins is unclear. An important cell-surface protein is CD4, the primary receptor of HIV-1. Here we show that mechanical force activates conformational and chemical changes on CD4 that may be important during viral attachment. We have used single-molecule force spectroscopy and analysis on HIV-1 infectivity to demonstrate that the mechanical extension of CD4 occurs in a time-dependent manner and correlates with HIV-1 infectivity. We show that Ibalizumab, a monoclonal antibody that blocks HIV-1, prevents the mechanical extension of CD4 domains 1 and 2. Furthermore, we demonstrate that thiol/disulfide exchange in CD4 requires force for exposure of cryptic disulfide bonds. This mechanical perspective provides unprecedented information that can change our understanding on how viruses interact with their hosts.