Mechanochemical Evolution of the Giant Muscle Protein Titin as Inferred from Ancient Proteins

Abstract

We use phylogenetic analysis and single-molecule atomic force spectroscopy (AFS) to investigate the nanomechanical evolution of titin, a giant protein responsible for the elasticity, integrity and signal transduction of muscle filaments. We bring back to life an eight-domain fragment of titin (I65-I72) from different extinct species ranging from 179 to 356 Mya that have been compared with their modern descendants. Using AFS techniques we have observed that the mechanical stability of titin domains and the presence of disulfide bonds are key elements in the evolution of titin. Our experiments together with structure prediction analysis and biochemical assays confirm that ancient titin domains were richer in disulfide bonds and have higher mechanical stability. While titin domains from birds and reptiles have kept similar mechanochemical features than those of the ancient ones, titin from mammals show lower mechanical stability and lower number of disulfide bonds. Our findings show a paleomechanical trend that suggests that disulfide bonds are mechanical regulators in titin that have disappeared over the course of evolution in mammals generating more flexible and extensible titin domains. We hypothesize that this substitution derived in physiological changes that allowed the muscular development and morphological diversity of modern vertebrates. A comparative analysis between animal physiological properties and titin mechanochemical properties supports our hypothesis and also allows us inferring some physiological properties of ancient species.