Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

Nature Structural & Molecular Biology volume 24, pages 652–657 (2017)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Reconstruction of ancestral titin fragments.
Figure 2: Single-molecule experiments with titin.
Figure 3: Mechanical stability versus geological time.
Figure 4: Force-clamp experiments for detection of disulfide-bond reductions catalyzed by Trx.
Figure 5: Correlation of mechanical stability with animal body mass.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Fürst, D.O., Osborn, M., Nave, R. & Weber, K. The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J. Cell Biol. 106, 1563–1572 (1988).

    Article  PubMed  Google Scholar 

  2. Labeit, S. & Kolmerer, B. Titins: giant proteins in charge of muscle ultrastructure and elasticity. Science 270, 293–296 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Rivas-Pardo, J.A. et al.Work done by titin protein folding assists muscle contraction. Cell Rep. 14, 1339–1347 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Erwin, D.H. et al. The Cambrian conundrum: early divergence and later ecological success in the early history of animals. Science 334, 1091–1097 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Alegre-Cebollada, J. et al. S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding. Cell 156, 1235–1246 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hall, B.G. Simple and accurate estimation of ancestral protein sequences. Proc. Natl. Acad. Sci. USA 103, 5431–5436 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Merkl, R. & Sterner, R. Ancestral protein reconstruction: techniques and applications. Biol. Chem. 397, 1–21 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Kratzer, J.T. et al. Evolutionary history and metabolic insights of ancient mammalian uricases. Proc. Natl. Acad. Sci. USA 111, 3763–3768 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zakas, P.M. et al. Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction. Nat. Biotechnol. 35, 35–37(2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gaucher, E.A., Govindarajan, S. & Ganesh, O.K. Palaeotemperature trend for Precambrian life inferred from resurrected proteins. Nature 451, 704–707 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Perez-Jimenez, R. et al. Single-molecule paleoenzymology probes the chemistry of resurrected enzymes. Nat. Struct. Mol. Biol. 18, 592–596 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Eick, G.N., Bridgham, J.T., Anderson, D.P., Harms, M.J. & Thornton, J.W. Robustness of reconstructed ancestral protein functions to statistical uncertainty. Mol. Biol. Evol. 34, 247–261 (2017).

    CAS  PubMed  Google Scholar 

  14. Hanson-Smith, V., Kolaczkowski, B. & Thornton, J.W. Robustness of ancestral sequence reconstruction to phylogenetic uncertainty. Mol. Biol. Evol. 27, 1988–1999 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Randall, R.N., Radford, C.E., Roof, K.A., Natarajan, D.K. & Gaucher, E.A. An experimental phylogeny to benchmark ancestral sequence reconstruction. Nat. Commun. 7, 12847 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hedges, S.B., Marin, J., Suleski, M., Paymer, M. & Kumar, S. Tree of life reveals clock-like speciation and diversification. Mol. Biol. Evol. 32, 835–845 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Benton, M.J. et al. Constraints on the timescale of animal evolutionary history. Palaeontologica Electronica 18.1, 18.1.1FC (2015).

    Google Scholar 

  18. Neagoe, C., Opitz, C.A., Makarenko, I. & Linke, W.A. Gigantic variety: expression patterns of titin isoforms in striated muscles and consequences for myofibrillar passive stiffness. J. Muscle Res. Cell Motil. 24, 175–189 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. von Castelmur, E. et al. A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain. Proc. Natl. Acad. Sci. USA 105, 1186–1191 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Watanabe, K., Muhle-Goll, C., Kellermayer, M.S., Labeit, S. & Granzier, H. Different molecular mechanics displayed by titin's constitutively and differentially expressed tandem Ig segments. J. Struct. Biol. 137, 248–258 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Dayhoff, M.O., Schwartz, R.M. & Orcutt, B.C. in Atlas of Protein Sequence and Structure Vol. 5, Suppl. 3 (National Biomedical Research Foundation, 1978).

  22. Wong, J.W., Ho, S.Y. & Hogg, P.J. Disulfide bond acquisition through eukaryotic protein evolution. Mol. Biol. Evol. 28, 327–334 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Kosuri, P. et al. Protein folding drives disulfide formation. Cell 151, 794–806 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Grützner, A. et al. Modulation of titin-based stiffness by disulfide bonding in the cardiac titin N2-B unique sequence. Biophys. J. 97, 825–834 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Mayans, O., Wuerges, J., Canela, S., Gautel, M. & Wilmanns, M. Structural evidence for a possible role of reversible disulphide bridge formation in the elasticity of the muscle protein titin. Structure 9, 331–340 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Li, H. & Fernandez, J.M. Mechanical design of the first proximal Ig domain of human cardiac titin revealed by single molecule force spectroscopy. J. Mol. Biol. 334, 75–86 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Perez-Jimenez, R., Garcia-Manyes, S., Ainavarapu, S.R. & Fernandez, J.M. Mechanical unfolding pathways of the enhanced yellow fluorescent protein revealed by single molecule force spectroscopy. J. Biol. Chem. 281, 40010–40014 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Ainavarapu, S.R. et al. Contour length and refolding rate of a small protein controlled by engineered disulfide bonds. Biophys. J. 92, 225–233 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Perez-Jimenez, R. et al. Diversity of chemical mechanisms in thioredoxin catalysis revealed by single-molecule force spectroscopy. Nat. Struct. Mol. Biol. 16, 890–896 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wiita, A.P. et al. Probing the chemistry of thioredoxin catalysis with force. Nature 450, 124–127 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Alegre-Cebollada, J., Kosuri, P., Rivas-Pardo, J.A. & Fernández, J.M. Direct observation of disulfide isomerization in a single protein. Nat. Chem. 3, 882–887 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Rogers, L.K., Leinweber, B.L. & Smith, C.V. Detection of reversible protein thiol modifications in tissues. Anal. Biochem. 358, 171–184 (2006).

    Article  CAS  PubMed  Google Scholar 

  33. Legendre, L.J., Guénard, G., Botha-Brink, J. & Cubo, J. Palaeohistological evidence for ancestral high metabolic rate in Archosaurs. Syst. Biol. 65, 989–996 (2016).

    Article  PubMed  Google Scholar 

  34. Lindstedt, S.L. & Schaeffer, P.J. Use of allometry in predicting anatomical and physiological parameters of mammals. Lab. Anim. 36, 1–19 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Burness, G.P., Leary, S.C., Hochachka, P.W. & Moyes, C.D. Allometric scaling of RNA, DNA, and enzyme levels: an intraspecific study. Am. J. Physiol. 277, R1164–R1170 (1999).

    CAS  PubMed  Google Scholar 

  36. Luo, Z.X. et al. Mammalian evolution. Evolutionary development in basal mammaliaforms as revealed by a docodontan. Science 347, 760–764 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Luo, Z.X., Yuan, C.X., Meng, Q.J. & Ji, Q. A Jurassic eutherian mammal and divergence of marsupials and placentals. Nature 476, 442–445 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Martin, T. et al. A Cretaceous eutriconodont and integument evolution in early mammals. Nature 526, 380–384 (2015).

    Article  CAS  PubMed  Google Scholar 

  39. Carroll, R.L. A Middle Pennsylvanian captorhinomorph, and the interrelationships of primitive reptiles. J. Paleo. 43, 151–170 (1969).

    Google Scholar 

  40. Bi, S., Wang, Y., Guan, J., Sheng, X. & Meng, J. Three new Jurassic euharamiyidan species reinforce early divergence of mammals. Nature 514, 579–584 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Sallan, L. & Galimberti, A.K. Body-size reduction in vertebrates following the end-Devonian mass extinction. Science 350, 812–815 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Paton, R.L., Smithson, T.R. & Clack, J.A. An amniote-like skeleton from the Early Carboniferous of Scotland. Nature 398, 508–513 (1999).

    Article  CAS  Google Scholar 

  43. O'Leary, M.A. et al. The placental mammal ancestor and the post-K-Pg radiation of placentals. Science 339, 662–667 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Abascal, F., Zardoya, R. & Posada, D. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105 (2005).

    Article  CAS  PubMed  Google Scholar 

  45. Drummond, A.J., Suchard, M.A., Xie, D. & Rambaut, A. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wilgenbusch, J.C. & Swofford, D. Inferring evolutionary trees with PAUP*. Curr. Protoc. Bioinformatics Chapter 6: Unit 6.4.1–6.4.28 (2003).

  47. Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586–1591 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Kahn, T.B., Fernández, J.M. & Perez-Jimenez, R. Monitoring oxidative folding of a single protein catalyzed by the disulfide oxidoreductase DsbA. J. Biol. Chem. 290, 14518–14527 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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. Fernández (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.

Author information

Authors and Affiliations

  1. CIC nanoGUNE, San Sebastian, Spain

    Aitor Manteca, Jörg Schönfelder, Alvaro Alonso-Caballero, Marie J Fertin, Nerea Barruetabeña, David De Sancho & Raul Perez-Jimenez

  2. Laboratory of Molecular Modeling, Federal University of São João del-Rei, São João del-Rei, Brazil

    Bruna F Faria

  3. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain

    Elias Herrero-Galán & Jorge Alegre-Cebollada

  4. IKERBASQUE, Basque Foundation for Science, Bilbao, Spain

    David De Sancho & Raul Perez-Jimenez

  5. Evolution and Genomics Technologies, S. L. (Evolgene), San Sebastian, Spain

    Raul Perez-Jimenez

Authors
  1. Aitor Manteca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jörg Schönfelder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alvaro Alonso-Caballero
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marie J Fertin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Nerea Barruetabeña
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bruna F Faria
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Elias Herrero-Galán
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jorge Alegre-Cebollada
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David De Sancho
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Raul Perez-Jimenez
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.P.-J. designed the research. A.M., B.F.F., N.B., D.D.S. and R.P.-J. conducted phylogenetic analysis. A.M. and M.J.F. cloned and expressed proteins. A.M., A.A.-C., J.S., D.D.S. and R.P.-J. performed AFM experiments and data analysis. E.H.-G. and J. A.-C. performed biochemical determination of disulfides in titin fragments. R.P-J. drafted the paper and all authors contributed in revising and editing the manuscript.

Corresponding author

Correspondence to Raul Perez-Jimenez.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Phylogenetic trees of titin

(a) Phylogenetic tree of titin using parsimony. A total of 33 titin molecules from different animals were used to reconstruct the phylogentic tree. Bootstrap support for each bifurcation is indicated. (b) Phylogenetic tree of titin using Bayesian inference. This tree contains new and updated sequences that appeared later during this study. A total of 37 titin molecules from different animals were used to reconstruct the phylogentic tree by Bayesian inference using MCMC. Posterior probabilities for each bifurcation are indicated. Different sources were considered for divergence times of ancestral nodes (Hedges, S. B. et al. Mol Biol Evol 32, 835-845, 2015 and Benton, M. J. et al.. Palaeontol Electronica 18, 1-106, 2015).

Supplementary Figure 2 Posterior probability distribution for each inferred residue of all ancestral titin fragments.

The residue with the highest posterior probability is assigned at each position.

Supplementary Figure 3 Phylogenetic tree used for the reconstruction of the ancestral myosin.

A total of 26 sequences were used. The tree was built by Bayesian inference using MCMC. The ancestors of interest are displayed with colored dots. We analyzed the sequences from LTCA (Last Tetrapod Common Ancestor), LSCA (Last Sauropod Common Ancestor) and LMCA (Last Mammal Common Ancestor) myosin molecules. Divergence times for ancestral nodes were collected from different sources (Hedges, S. B. et al. Mol Biol Evol 32, 835-845, 2015 and Benton, M. J. et al.. Palaeontol Electronica 18, 1-106, 2015). Posterior probabilities for each bifurcation are indicated.

Supplementary Figure 4 Mutational analysis of titin and myosin.

Mutation rates for titin (a) and myosin (b) are estimated as the number of mutations from the ancestral forms to their modern counterparts per 100 residues and per Myr. Overall, mutation rate is double in titin with respect to myosin. Mutation rates for titin and myosin are estimated as the number of mutations from the ancestral forms to their modern counterparts per 100 residues and per Myr. Overall, mutation rate is double in titin (grey bars) with respect to myosin (white bars). Analysis of residue replacement and mutability for LTCA and human titin proximal (c) and distal (d) I-band, A-band (e) and myosin (f). For each residue type we estimate the occurrence in the different titin fragment as well as myosin for human and LTCA titin. The difference is the replacement for each residue type in the transition between LTCA and human titin. We thus estimate the ratio between replacement and relative mutability considering Ala as reference with a value of 100 (Dayhoff M.O. et al. Biomedical Research Foundation, Silver Spring: MD, 1978; Vol. 5. 3). In the plot this ratio is represented for each residue. A positive value (red bars) indicate an increasing number of the specific amino acid in the transition from LTCA to human. The blue bars represent a decreasing number of residues. Cysteine residues have decreased their representation in the I-band of human titin much more prominently than any other residue. In the contrary, these residues have increased their number in the A-band of titin and myosin.

Supplementary Figure 5 Scatter plots of contour lengths and unfolding forces for all the titin fragments studied, with kernel density estimates shown as lines.

Data collection for each protein is n=374 for LTCA, n=614 for LSCA, n= 407 for LMCA, n= 366 for LPMCA, n= 409 for zebra finch, n= 375 for chicken, n= 263 for orca, n= 341 for rat and n= 347 for human titin fragment. Blue bars indicate force and length of domains with no disulfide bonds. Red bars represent domains with disulfide bridges. Domains that do not contain disulfide bonds and are thus fully extended are represented in blue, whereas those showing disulfide bonds are represented in red and display lower mechanical resistance. In the last graph, the average unfolding forces of ancestral and extant species are shown with square and circles, respectively. Unfolding forces in both types of domains increase when the percentage of experimental disulfide bonds is higher, following a linear relationship.

Supplementary Figure 6 Force-clamp experiment for detection of single disulfide reduction events

(a) Schematics of the experiment. Disulfide bonded domains (red) show a 2 two-step unfolding pattern. The first step (~12 nm) corresponds to the unfolding of the beta sheets that are not trapped in the disulfide bond while the second step (~15 nm) shows the unfolding of the rest of the protein after the reduction of the disulfide bond cause by thioredoxin. Not disulfide bonded domains have a single-step (~27 nm) unfolding pattern that represents the stretching of the whole domain. (b) Experimental trace in the absence of thioredoxin enzymes of LSCA titin with seven steps representing each immunoglobulin domain visualized within the first pulse at 135 pN (grey). The fully unfolded domains are marked with an arrow (~27 nm) whereas disulfide bonded domains are marked with an asterisk (~5-20 nm). No steps are detected in the second stage at 80 pN (green) that was kept for 20 s. (c) Two populations of steps can be observed in the histogram (n=286). The histogram below shows step captured in the 80 pN pulse where reduction are generally observed in the presence of Trx (Fig. 3 in main text). (d) and (e) Experimental trace and steps histograms (n=132) for human titin, respectively. In this case eight unfolding events are shown from which only one shows step size corresponding to a disulfide bonded domain. In the traces from human titin is common to observe 1 or 2 disulfide bonded domains. In both cases the histograms of the unfolding pulse resemble the histograms of the force-extension experiments, as expected.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 1609 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Manteca, A., Schönfelder, J., Alonso-Caballero, A. et al. Mechanochemical evolution of the giant muscle protein titin as inferred from resurrected proteins. Nat Struct Mol Biol 24, 652–657 (2017). https://doi.org/10.1038/nsmb.3426

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3426

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing