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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. ; Sí

Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a long-standing goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can "resurrect" efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30–90 °C), pH values (4–10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome. ; We thank Prof. Ed Bayer's group for kindly providing the plasmids used in the minicellulosome constructs. Research was supported by the Basque Government grant ELKARTEK to R.P.-J, and also partly by Ministry of Economy and Competitiveness (MINECO) grant BIO2016-77390-R, BFU2015-71964 to R.P.-J., BIO2016-74875-P to J. A.G., and CTQ2015-65320-R and RYC-2016-19590 to D.D.S.; European Commission grant CIG Marie Curie Reintegration program FP7-PEOPLE-2014 to R.P.-J, and European Commission grant NMP-FP7 604530-2 (CellulosomePlus), and the ERA-IB EIB.12.022 grant (FiberFuel) funded by the MINECO (PCIN-2013-011-C02-01) to M.C.-V. We also thank Fundación Repsol and Gipuzkoako Foru Aldundia for financial support.