Elasticity of the Transition State Leading to an Unexpected Mechanical Stabilization of Titin Immunoglobulin Domains

Abstract

The giant protein titin plays a critical role in regulating the passive elasticity of muscles, mainly through the stochastic unfolding and refolding of its numerous immunoglobulin domains in the I-band of sarcomeres. The unfolding dynamics of titin immunoglobulin domains at a force range greater than 100 pN has been studied by atomic force microscopy, while that at smaller physiological forces has not been measured before. By using magnetic tweezers, it is found that the titin I27 domain unfolds in a surprising non-monotonic force-dependent manner at forces smaller than 100 pN, with the slowest unfolding rate occurring around 22 pN. We further demonstrate that a model with single unfolding pathway taking into account the elasticity of the transition state can reproduce the experimental results. These results provide important novel insights into the regulation mechanism of the passive elasticity of muscle tissues.

Proteins Breaking Bad: A Free Energy Perspective

“…In particular, understanding the elastic properties of tandem modular proteins within this context supposes a highly non-trivial enterprise 24–25 . A rigorous description on that level should convolute the folding properties of the domains with the non-hookean response of the polypeptide chain to force 26–28 .…”

Phosphorylation Reduces the Mechanical Stability of the α‐Catenin/ β‐Catenin Complex

“…Utilizing a single‐molecule construct assay for interprotein interactions, we designed a single‐molecule construct for direct and efficient quantification of the mechanical stability of the α‐catenin/β‐catenin complex and the effects of phosphorylation. In the single‐molecule construct, the αN12 (the N1 and N2 domains of α‐catenin) and βNt are linked through a long, flexible, unstructured polypeptide chain (196 amino acids), and are spanned between four titin Ig 27 th domain (I27) . The N‐ and C‐ termini of the construct contain a biotinylated avi‐tag and a spy‐tag, respectively, for specific tethering (Figure ).…”

“…The four repeats of the well‐characterized I27 domain (two repeats at each end) of the essential component act as a molecular spacer. The I27 domain is highly mechanically stable and it unfolds with an unfolding rate of approximately 10 −3 s −1 over the force range of 2–20 pN . Furthermore, there is a 572‐bp DNA handle added between the single‐molecule construct and the super‐paramagnetic bead as additional spacer to avoid non‐specific interaction between the bead and surface.…”

The extracellular matrix–myosin pathway in mechanotransduction: from molecule to tissue

“…When catch-bonds form, there is an initial increase with force of the barrier height separating the folded and unfolded states. Further increase in force will eventually drive the system to slip-bond kinetics, resulting in a non-monotonic catch-to-slip switching behavior [9,10]. Second, force shifts the position of the folded and unfolded minima along the energy and extension coordinates.…”

“…When repeatedly exposing a single protein to a given force, the same molecule takes a slightly different time to transition from the folded to the unfolded state. This variation in the dwell time can be explained by the multidimensionality of the folding energy landscape of a protein [10,13]. This non-deterministic behavior means that, while less frequent, proteins will still unfold at low or zero forces, while this process, in the absence of catchbonds, will be more frequent as force increases [14].…”