Determining Stable Single Alpha Helical (SAH) Domain Properties by Circular Dichroism and Atomic Force Microscopy

  • Matthew Batchelor
  • Marcin Wolny
  • Marta Kurzawa
  • Lorna Dougan
  • Peter J. Knight
  • Michelle Peckham
Part of the Methods in Molecular Biology book series (MIMB, volume 1805)


Stable, single α-helical (SAH) domains exist in a number of unconventional myosin isoforms, as well as other proteins. These domains are formed from sequences rich in charged residues (Arg, Lys, and Glu), they can be hundreds of residues long, and in isolation they can tolerate significant changes in pH and salt concentration without loss in helicity. Here we describe methods for the preparation and purification of SAH domains and SAH domain-containing constructs, using the myosin 10 SAH domain as an example. We go on to describe the use of circular dichroism spectroscopy and force spectroscopy with the atomic force microscope for the elucidation of structural and mechanical properties of these unusual helical species.

Key words

Stable single alpha helical domains Circular dichroism Atomic force microscopy Myosin 



This work was supported by Biotechnology and Biological Sciences Research Council grants BB/I007423/1 and BB/M009114/1.


  1. 1.
    Knight PJ, Thirumurugan K, Xu Y, Wang F, Kalverda AP, Stafford WF III, Sellers JR, Peckham M, (2005) The Predicted Coiled-coil Domain of Myosin 10 Forms a Novel Elongated Domain That Lengthens the Head. J Biol Chem 280:34702–34708CrossRefPubMedGoogle Scholar
  2. 2.
    Spink BJ, Sivaramakrishnan S, Lipfert J, Doniach S, Spudich JA (2008) Long single alpha-helical tail domains bridge the gap between structure and function of myosin VI. Nat Struct Mol Biol 15:591–597CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Baboolal TG, Sakamoto T, Forgacs E, White HD, Jackson SM, Takagi Y, Farrow RE, Molloy JE, Knight PJ, Sellers JR, Peckham M (2009) The SAH domain extends the functional length of the myosin lever. Proc Natl Acad Sci U S A 106:22193–22198CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Li J, Chen Y, Deng Y, Unarta IC, Lu Q, Huang X, Zhang M (2017) Ca2+-induced rigidity change of the myosin VIIa IQ motif-single α helix lever arm extension. Structure 25:579–591Google Scholar
  5. 5.
    Yang Y, Baboolal TG, Siththanandan V, Chen M, Walker ML, Knight PJ, Peckham M, Sellers JR (2009) A FERM domain autoregulates Drosophila myosin 7a activity. Proc Natl Acad Sci U S A 106:4189–4194CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Peckham M, Knight PJ (2009) When a predicted coiled coil is really a single α-helix, in myosins and other proteins. Soft Matter 5:2493–2503Google Scholar
  7. 7.
    Wolny M, Batchelor M, Knight PJ, Paci E, Dougan L, Peckham M (2014) Stable single α-helices are constant force springs in proteins. J Biol Chem 289:27825–27835CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Wang CL, Chalovich JM, Graceffa P, Lu RC, Mabuchi K, Stafford W (1991) A long helix from the central region of smooth muscle caldesmon. J Biol Chem 266:13958–13963Google Scholar
  9. 9.
    Sivaramakrishnan S, Sung J, Ali M, Doniach S, Flyvbjerg H, Spudich JA (2009) Combining single-molecule optical trapping and small-angle X-ray scattering measurements to compute the persistence length of a protein ER/K α-helix. Biophys J 97:2993–2999Google Scholar
  10. 10.
    Samejima K, Platani M, Wolny M, Ogawa H, Vargiu G, Knight PJ, Peckham M, Earnshaw WC (2015) The inner centromere protein (INCENP) coil is a single α-helix (SAH) domain that binds directly to microtubules and is important for chromosome passenger complex (CPC) localization and function in mitosis. J Biol Chem 290:21460–21472Google Scholar
  11. 11.
    Wolny M, Batchelor M, Bartlett GJ, Baker EG, Kurzawa M, Knight PJ, Dougan L, Woolfson DN, Paci E, Peckham M (2017) Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability. Sci Rep 7:44341Google Scholar
  12. 12.
    Greenfield NJ (2006) Using circular dichroism spectra to estimate protein secondary structure. Nat Protoc 1:2876–2890CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Greenfield NJ (2006) Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Protoc 1:2527–2535CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Wolny M, Colegrave M, Colman L, White E, Knight PJ, Peckham M (2013) Cardiomyopathy mutations in the tail of β-cardiac myosin modify the coiled-coil structure and affect integration into thick filaments in muscle sarcomeres in adult cardiomyocytes. J Biol Chem 288:31952–31962Google Scholar
  15. 15.
    Mitsui K, Hara M, Ikai A (1996) Mechanical unfolding of α2-macroglobulin molecules with atomic force microscope. FEBS Lett 385:29–33CrossRefPubMedGoogle Scholar
  16. 16.
    Rief M, Gautel M, Oesterhelt F, Fernandez JM, Gaub HE (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276:1109–1112CrossRefPubMedGoogle Scholar
  17. 17.
    Hoffmann T, Dougan L (2012) Single molecule force spectroscopy using polyproteins. Chem Soc Rev 41:4781–4796CrossRefPubMedGoogle Scholar
  18. 18.
    Best RB, Li B, Steward A, Daggett V, Clarke J (2001) Can non-mechanical proteins withstand force? Stretching barnase by atomic force microscopy and molecular dynamics simulation. Biophys J 81:2344–2356CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li H, Oberhauser AF, Redick SD, Carrion-Vazquez M, Erickson HP, Fernandez JM (2001) Multiple conformations of PEVK proteins detected by single-molecule techniques. Proc Natl Acad Sci U S A 98:10682–10686CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Oroz J, Hervas R, Valbuena A, Carrion-Vazquez M (2012) Unequivocal single-molecule force spectroscopy of intrinsically disordered proteins. Methods Mol Biol 896:71–87PubMedGoogle Scholar
  21. 21.
    Tskhovrebova L, Trinick J, Sleep JA, Simmons RM (1997) Elasticity and unfolding of single molecules of the giant muscle protein titin. Nature 387:308–312CrossRefPubMedGoogle Scholar
  22. 22.
    Carrion-Vazquez M, Oberhauser AF, Fowler SB, Marszalek PE, Broedel SE, Clarke J, Fernandez JM (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci U S A 96:3694–3699CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Improta S, Politou AS, Pastore A (1996) Immunoglobulin-like modules from titin I-band: extensible components of muscle elasticity. Structure 4:323–337CrossRefPubMedGoogle Scholar
  24. 24.
    Brockwell DJ, Beddard GS, Clarkson J, Zinober RC, Blake AW, Trinick J, Olmsted PD, Smith DA, Radford SE (2002) The effect of core destabilization on the mechanical resistance of I27. Biophys J 83:458–472CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Muller S, Hoege C, Pyrowolakis G, Jentsch S (2001) SUMO, ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol 2:202–210CrossRefPubMedGoogle Scholar
  26. 26.
    Zinober RC, Brockwell DJ, Beddard GS, Blake AW, Olmsted PD, Radford SE, Smith DA (2002) Mechanically unfolding proteins: the effect of unfolding history and the supramolecular scaffold. Protein Sci 11:2759–2765CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lobley A, Whitmore L, Wallace BA (2002) DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 18:211–212CrossRefPubMedGoogle Scholar
  28. 28.
    Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:W668–W673CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Kelly SM, Jess TJ, Price NC (2005) How to study proteins by circular dichroism. Biochim Biophys Acta 1751:119–139CrossRefPubMedGoogle Scholar
  30. 30.
    Greenfield N, Fasman GD (1969) Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8:4108–4116CrossRefPubMedGoogle Scholar
  31. 31.
    Chen YH, Yang JT, Chau KH (1974) Determination of the helix and β form of proteins in aqueous solution by circular dichroism. Biochemistry 13:3350–3359CrossRefPubMedGoogle Scholar
  32. 32.
    Woody RW (1995) Circular dichroism. Methods Enzymol 246:34–71CrossRefPubMedGoogle Scholar
  33. 33.
    Law R, Carl P, Harper S, Dalhaimer P, Speicher DW, Discher DE (2003) Cooperativity in forced unfolding of tandem spectrin repeats. Biophys J 84:533–544CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Hutter J, Bechhoefer J (1993) Calibration of atomic-force microscope tips. Rev Sci Instrum 64:1868–1873CrossRefGoogle Scholar
  35. 35.
    Cappella B, Dietler G (1999) Force-distance curves by atomic force microscopy. Surf Sci Rep 34:1–104CrossRefGoogle Scholar
  36. 36.
    Rounsevell R, Forman JR, Clarke J (2004) Atomic force microscopy: mechanical unfolding of proteins. Methods 34:100–111CrossRefPubMedGoogle Scholar
  37. 37.
    Zocher M, Zhang C, Rasmussen SG, Kobilka BK, Muller DJ (2012) Cholesterol increases kinetic, energetic, and mechanical stability of the human β2-adrenergic receptor. Proc Natl Acad Sci U S A 109:E3463–E3472CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Leitner M, Fantner GE, Fantner EJ, Ivanova K, Ivanov T, Rangelow I, Ebner A, Rangl M, Tang J, Hinterdorfer P (2012) Increased imaging speed and force sensitivity for bio-applications with small cantilevers using a conventional AFM setup. Micron 43:1399–1407CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Berkemeier F, Bertz M, Xiao S, Pinotsis N, Wilmanns M, Grater F, Rief M (2011) Fast-folding α-helices as reversible strain absorbers in the muscle protein myomesin. Proc Natl Acad Sci U S A 108:14139–14144CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefPubMedGoogle Scholar
  41. 41.
    Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948–1954CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Matthew Batchelor
    • 1
  • Marcin Wolny
    • 1
  • Marta Kurzawa
    • 1
  • Lorna Dougan
    • 2
  • Peter J. Knight
    • 1
  • Michelle Peckham
    • 1
  1. 1.Astbury Centre for Structural Molecular Biology and School of Molecular and Cellular Biology, Faculty of Biological SciencesUniversity of LeedsLeedsUK
  2. 2.Astbury Centre for Structural Molecular Biology and School of Physics and AstronomyUniversity of LeedsLeedsUK

Personalised recommendations