Abstract
Disulfide bonds play a pivotal role in the mechanical stability of proteins. Numerous proteins that are known to be exposed to mechanical forces in vivo contain disulfide bonds. The presence of cryptic disulfide bonds in a protein structure may be related to its resistance to an applied mechanical force. Disulfide bonds in proteins tend to be highly conserved but their evolution might be directly related to the evolution of the protein mechanical stability. Hence, tracking the evolution of disulfide bonds in a protein can help to derive crucial stability/function correlations in proteins that are exposed to mechanical forces. Phylogenic analysis and ancestral sequence reconstruction (ASR) allow tracking the evolution of proteins from the past ancestors to our modern days and also establish correlations between proteins from different species. In addition, ASR can be combined with single-molecule force spectroscopy (smFS) to investigate the mechanical properties of proteins including the occurrence and function of disulfide bonds. Here we present a detailed protocol to study the mechanochemical evolution of proteins using a fragment of the giant muscle protein titin as example. The protocol can be easily adapted to AFS studies of any resurrected mechanical force bearing protein of interest.
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Acknowledgments
A.A.-C. is funded by the predoctoral program of the Basque Government. We acknowledge financial support from the Spanish Ministry of Economy, Industry and Competitiveness grant BIO2016-77390-R to R. P.-J. and Marie Curie Career Integration Grants (CIG) FP7-PEOPLE-2013-CIG from the European Commission to R. P.-J. This work was also supported by the Spanish Ministry of Economy, Industry and Competitiveness under the Maria de Maeztu Units of Excellence Program - MDM-2016-0618.
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Schönfelder, J., Alonso-Caballero, A., Perez-Jimenez, R. (2022). Mechanochemical Evolution of Disulfide Bonds in Proteins. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_15
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DOI: https://doi.org/10.1007/978-1-0716-1716-8_15
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