Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins

Abstract

The sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this diversity and its evolution has not been proposed. We use phylogenetic analyses and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We resurrect eight-domain fragments of titin corresponding to the common ancestors to mammals, sauropsids, and tetrapods, which lived 105-356 Myr ago, and compare them with titin fragments from some of their modern descendants. We demonstrate that the resurrected titin molecules are rich in disulfide bonds and display high mechanical stability. These mechanochemical elements have changed over time, creating a paleomechanical trend that seems to correlate with animal body size, allowing us to estimate the sizes of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.

Conserved cysteines in titin sustain the mechanical function of cardiomyocytes

“…In vitro, conserved cysteines B, F and G can be induced to form disulfide bonds that alter the nanomechanics of the parent Ig domains (21,24). Hence, a plausible scenario is that equivalent disulfides are present naturally in native titin as mechanical rheostats.…”

“…The N2Bus region also contains cysteines that can stiffen titin through disulfide bond formation (23), albeit they are less evolutionary conserved than Ig domain cysteines (53% mean conservation, Figure 1F, Supplementary Figure S1C). Beyond the remarkable conservation of I-band titin's cysteine residues and their role in the evolution of the protein in vertebrates (24), the in vivo relevance of redox mechanical modulation of titin is supported by limited data on the global oxidation of the protein (25)(26)(27)(28), the effects of redox-active molecules on striated muscle mechanics (22,23,29,30), and the disulfide-compatible location of the majority of structurally conserved cysteines of titin (21,31).…”

“…The longest currently available titin sequences for the 37 species reported in (24) were aligned using Clustal Omega, with the only exception of chicken, for which no titin sequence with enough coverage was found in Uniprot, GenomeNet or NCBI databases. Sequence identification codes are listed in Supplementary Note S3.…”

Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

“…In the past decade or so, the so-called ancestral sequence resurrection technique (ASR) has been used to study the evolution of genes and proteins [10][11][12] . ASR utilizes sequences of proteins or genes from different species to create phylogenetic relationships, from which the sequences of their ancestors can be predicted and reconstructed in the laboratory 13 .…”

“…Using a diverse combination of sequences, it is even possible to reconstruct Precambrian proteins belonging to organisms that lived shortly after the origin of life 10,11 . Reconstructed ancestral proteins have displayed enhanced thermal or mechanical stability, better pH response, improved activity and expression level, chemical promiscuity, and in some cases, all of these at once [10][11][12]14 . These traits are thought to reflect the conditions in which these ancestral proteins lived.…”