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

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.Research has been supported by the Ministry of Economy and Competitiveness (MINECO) grant BIO2016-77390-R, BFU2015-71964 to R.P.-J., BIO2014-54768-P and RYC-2014-16604 to J.A-C., and CTQ2015-65320-R to D.D.S., and the European Commission grant CIG Marie Curie Reintegration program FP7-PEOPLE-2014 to R.P.-J. A.A.-C. is funded by the predoctoral program of the Basque Government. R.P.-J. and D.D.S., thank CIC nanoGUNE and the Ikerbasque Foundation for Science for financial support. CNIC is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Plasmid pQE80-(I91-32/75)8 was a kind gift from J. Fernandez (Columbia University). We thank R. Zardoya (National Museum of Natural Sciences, Madrid) for helpful discussions and comments. The authors acknowledge technical support provided by IZO-SGI SGIker of UPV/EHU and European funding (ERDF and ESF) for the use of the Arina HPC cluster and the assistance provided by T. Mercero and E. Ogando.S

Single-molecule paleoenzymology probes the chemistry of resurrected enzymes

A journey back in time is possible at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx), dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32° C more stable than modern enzymes and the oldest show significantly higher activity than extant ones at pH 5. We probed their mechanisms of reduction using single-molecule force spectroscopy. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs utilize chemical mechanisms of reduction similar to those of modern enzymes. While Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over a 4 Gyr time span to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth