Mechanochemical evolution of the giant muscle protein tititn as inferred from resurrecter proteins

161 p.The sarcomere-based structure of muscles is conserved among vertebrates; however, vertebrate muscle physiology is extremely diverse. A molecular explanation for this muscle diversity and its evolution has not been proposed. In this Thesis, We use phylogenetic analysis and single-molecule force spectroscopy (smFS) to investigate the mechanochemical evolution of titin, a giant protein responsible for the elasticity of muscle filaments. We bring back to life eight-domain fragments of titin corresponding to ancestors to mammals, sauropsids, and tetrapods, that lived 105-356 Myr, and compare them with some of their modern descendants. We demonstrate that 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 correlates with animal body size, allowing us to estimate the size of extinct species. We hypothesize that mechanical adjustments in titin contributed to physiological changes that allowed the muscular development and diversity of modern tetrapods.CICNanoGUN

In vitro Production of Hemin-Based Artificial Metalloenzymes

Developing enzyme alternatives is pivotal to improving and enabling new processes in biotechnology and industry. Artificial metalloenzymes (ArMs) are combinations of protein scaffolds with metal elements, such as metal nanoclusters or metal-containing molecules with specific catalytic properties, which can be customized. Here, we engineered an ArM based on the consensus tetratricopeptide repeat (CTPR) scaffold by introducing a unique histidine residue to coordinate the hemin cofactor. Our results show that this engineered system exhibits robust peroxidase-like catalytic activity driven by the hemin. The expression of the scaffold and subsequent coordination of hemin was achieved by recombinant expression in bulk and through in vitro transcription and translation systems in water-in-oil drops. The ability to synthesize this system in emulsio paves the way to improve its properties by means of droplet microfluidic screenings, facilitating the exploration of the protein combinatorial space to discover improved or novel catalytic activities.A.M. is financed by grant 2022-FELL-000011-01 funded by Gipuzkoa Fellows Program (Diputación Foral de Gipuzkoa) and grant “EPINPOC” co-funded by AECT Euroregion New Aquitaine-Navarra-Basque Country. A.L.C. acknowledges financial support by the Agencia Estatal de Investigación, Grants: PID2019-111649RB−I00 and PID2022-137977OB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and Grant PDC2021-120957-I00 funded by MCIN/AEI/ 10.13039/501100011033 and by the “European Union NextGenerationEU/PRTR”. A.L.C. also acknowledges financial support from Diputación Foral de Gipuzkoa grant 2023-QUAN-000023-01. This work was performed under the Maria de Maeztu Units of Excellence Program from Q5 the Spanish State Research Agency grant no. MDM-2017-0720. A.B. gratefully acknowledges the financial support from the Spanish Research Agency (AEI) for the financial support (PID2019-110239RB−I00 and PID2022-142128NB-I00 funded by MCIN/ AEI/10.13039/501100011033/ and by the ”European Union NextGenerationEU/PRTR”; RYC2018-025923-I from RyC program - MCIN/ AEI /10.13039/501100011033 and FSE “invierte en tu futuro”), BBVA Foundation - IN[21]_CBB_QUI_0086, and UPV/EHU- GIU21-033). We thank Thomas Beneyton and J−C Baret for their relentless support with the device fabrication and the mounting of the optical setup

Directed Evolution in Drops: Molecular Aspects and Applications

International audienceThe process of optimizing the properties of biological molecules is paramount for many industrial and medical applications. Directed evolution is a powerful technique for modifying and improving biomolecules such as proteins or nucleic acids (DNA or RNA). Mimicking the mechanism of natural evolution, one can enhance a desired property by applying a suitable selection pressure and sorting improved variants. Droplet-based microfluidic systems offer a high-throughput solution to this approach by helping to overcome the limiting screening steps and allowing the analysis of variants within increasingly complex libraries. Here, we review cases where successful evolution of biomolecules was achieved using droplet-based microfluidics, focusing on the molecular processes involved and the incorporation of microfluidics to the workflow. We highlight the advantages and limitations of these microfluidic systems compared to lowthroughput methods and show how the integration of these systems into directed evolution workflows can open new avenues to discover or improve biomolecules according to user-defined conditions

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