Structure-Based, In Silico Approaches for the Development of Novel cAMP FRET Reporters

  • Matías Machado
  • Sergio PantanoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1294)


A significant contribution to the research in cAMP signaling has been made by the development of genetically encoded FRET sensors that allow detection of local concentrations of second messengers in living cells. Nowadays, the availability of a number of 3D structures of cyclic nucleotide-binding domains (CNBD) undergoing conformational transitions upon cAMP binding, along with computational tools, can be exploited for the design of novel or improved sensors. In this chapter we will overview some coarse-grained geometrical considerations on fluorescent proteins, CNBD, and linker peptides to draw simple qualitative rules that may aid the design of novel sensors. Finally, we will illustrate how the application of these simple rules can be used to describe the mechanistic basis of cAMP sensors reported in the literature.


Fluorescent protein Allosteric mechanism CNBD Rational design Protein engineering Coarse grain SIRAH 



This work was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11, and Intramural Transversal Program 2013, Institut Pasteur de Montevideo. M.M and S.P are members of the SNI, ANII, Uruguay.


  1. 1.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefPubMedGoogle Scholar
  2. 2.
    Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759–781CrossRefPubMedGoogle Scholar
  3. 3.
    Newman RH, Fosbrink MD, Zhang J (2011) Genetically encodable fluorescent biosensors for tracking signaling dynamics in living cells. Chem Rev 111:3614–3666CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721CrossRefPubMedGoogle Scholar
  5. 5.
    Day RN, Davidson MW (2009) The fluorescent protein palette: tools for cellular imaging. Chem Soc Rev 38:2887–2921CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Lakowicz JR (1999) Energy transfer. Fluorescence spectroscopy. Kluwer Academic/Plenum, New York, pp 368–391Google Scholar
  7. 7.
    Sipieter F, Vandame P, Spriet C et al (2013) From FRET imaging to practical methodology for kinase activity sensing in living cells. Prog Mol Biol Transl Sci 113:145–216CrossRefPubMedGoogle Scholar
  8. 8.
    Hsin J, Arkhipov A, Yin Y et al. (2008) Using VMD: an introductory tutorial. Curr Protoc Bioinformat Chapter 5, Unit 5.7Google Scholar
  9. 9.
    Taylor SS, Ilouz R, Zhang P et al (2012) Assembly of allosteric macromolecular switches: lessons from PKA. Nat Rev Mol Cell Biol 13:646–658CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Zhang P, Smith-Nguyen EV, Keshwani MM et al (2012) Structure and allostery of the PKA RIIbeta tetrameric holoenzyme. Science 335:712–716CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Berman HM, Ten Eyck LF, Goodsell DS et al (2005) The cAMP binding domain: an ancient signaling module. Proc Natl Acad Sci U S A 102:45–50CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Rehmann H, Arias-Palomo E, Hadders MA et al (2008) Structure of Epac2 in complex with a cyclic AMP analogue and RAP1B. Nature 455:124–127CrossRefPubMedGoogle Scholar
  13. 13.
    Berrera M, Pantano S, Carloni P (2007) Catabolite activator protein in aqueous solution: a molecular simulation study. J Phys Chem B 111:1496–1501CrossRefPubMedGoogle Scholar
  14. 14.
    Berrera M, Pantano S, Carloni P (2006) cAMP Modulation of the cytoplasmic domain in the HCN2 channel investigated by molecular simulations. Biophys J 90:3428–3433CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Pantano S, Zaccolo M, Carloni P (2005) Molecular basis of the allosteric mechanism of cAMP in the regulatory PKA subunit. FEBS Lett 579:2679–2685CrossRefPubMedGoogle Scholar
  16. 16.
    Pantano S (2008) In silico description of fluorescent probes in vivo. J Mol Graph Model 27:563–567CrossRefPubMedGoogle Scholar
  17. 17.
    Nikolaev VO, Bunemann M, Hein L et al (2004) Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218CrossRefPubMedGoogle Scholar
  18. 18.
    Ansbacher T, Srivastava HK, Stein T et al (2012) Calculation of transition dipole moment in fluorescent proteins–towards efficient energy transfer. Phys Chem Chem Phys 14:4109–4117CrossRefPubMedGoogle Scholar
  19. 19.
    Topell S, Glockshuber R (2002) Circular permutation of the green fluorescent protein. Methods Mol Biol 183:31–48PubMedGoogle Scholar
  20. 20.
    Rief M, Oesterhelt F, Heymann B et al (1997) Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy. Science 275:1295–1297CrossRefPubMedGoogle Scholar
  21. 21.
    Carrion-Vazquez M, Oberhauser AF, Fowler SB et al (1999) Mechanical and chemical unfolding of a single protein: a comparison. Proc Natl Acad Sci U S A 96:3694–3699CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Flory PJ (1975) Spatial configuration of macromolecular chains. Science 188:1268–1276CrossRefPubMedGoogle Scholar
  23. 23.
    Di BG, Zoccarato A, Lissandron V et al (2008) Protein kinase A type I and type II define distinct intracellular signaling compartments. Circ Res 103:836–844CrossRefGoogle Scholar
  24. 24.
    Zaccolo M, De GF, Cho CY et al (2000) A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2:25–29CrossRefPubMedGoogle Scholar
  25. 25.
    Diller TC, Madhusudan, Xuong NH et al. (2001) Molecular basis for regulatory subunit diversity in cAMP-dependent protein kinase: crystal structure of the type II beta regulatory subunit. Structure 9, 73–82Google Scholar
  26. 26.
    Smith FD, Reichow SL, Esseltine JL et al (2013) Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation. Elife 2:e01319PubMedCentralPubMedGoogle Scholar
  27. 27.
    Lissandron V, Terrin A, Collini M et al (2005) Improvement of a FRET-based indicator for cAMP by linker design and stabilization of donor-acceptor interaction. J Mol Biol 354:546–555CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Group of Biomolecular SimulationsInstitut Pasteur de MontevideoMontevideoUruguay

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