Direct Observation of Titin Immunoglobulin Domain Unfolding-Refolding in Muscle Sarcomeres

Abstract

Titin is the largest protein in the human body. Constituted by Immunoglobulin-like (Ig) domains and unstructured regions, titin determines the passive elasticity of muscle tissue. Current models of muscle elasticity propose a central role for the extension of its unstructured regions at low force, while relegating Ig domains unfolding reactions as a safety mechanism triggered only by un-physiologically high forces. Here we combine single-particle tracking and single-molecule force spectroscopy to observe the dynamics of Ig domains from I-band under physiological forces. Using quantum dots and centroid tracking techniques we show that in single myofibrils extended to an optimal sarcomere length of 3.1 μm, titin molecules labeled in situ undergo stepwise changes in length of 13 ± 3 and 22 ± 3 nm. Additionally, single-molecule experiments on a proximal native fragment of titin shows that the folding/unfolding step sizes of the proximal Ig domains of 10.4 ± 3.6 and 21.8 ± 4.7 nm, at 8 pN of force. These extensions closely match the distribution of step sizes observed in the intact single myofibril. Our studies demonstrate that Ig domain folding reactions in titin occur at physiological forces and sarcomere lengths and are thus likely to be a major component of muscle elasticity.