Intermolecular Misfolding Captured in Parallelly Organized Titin

The giant muscle protein titin is largely responsible for the passive elasticity of the muscles. The I-band part of titin is elastic, and its constitutive immunoglobulin (Ig) domains undergo force-induced unfolding and refolding when the muscle is stretched toward or beyond the end of the physiological range of sarcomere length. Correct folding of the titin Ig domains is essential to the structure and functions of titin. Although our knowledge of titin elasticity at the molecular level has been largely obtained from single molecule experiments, titin does not exist as an isolated molecule. Instead, six titins are parallelly organized in the muscle sarcomeres. It remains unknown what impact such a parallel organization brings on the folding of titin Ig domains and titin elasticity. Using the two-molecule force spectroscopy technique, here, we report the direct observation of the intermolecular misfolding of titin Ig domains that are arranged in parallel. Our results reveal that when parallelly arranged, two I94 domains can misfold into an intermolecular domain-swapped state that is thermally and mechanically stable. Such intermolecular misfolding may play important structural and functional roles in titin organization and elasticity.