Mapping Calcium-Sensitive Regions in GCAPs by Site-Specific Fluorescence Labelling

  • Karl-Wilhelm KochEmail author
  • Jens Christoffers
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


Signal transduction processes that are under control of changes in cytoplasmic Ca2+-concentration involve Ca2+-sensor proteins, which often undergo pronounced conformational transitions triggered by Ca2+. Consequences of conformational changes can be the structural rearrangement of single amino acids, exposition of small patches of several amino acids, or the movement of whole protein regions or domains. Furthermore, these conformational changes can lead to the exposure or movement of posttranslationally attached acyl groups. These processes could then control the function of target proteins, for example, by Ca2+-dependent protein–protein interaction. Fluorescence spectroscopy allows for mapping these Ca2+-sensitive regions but needs site-specific fluorescence labelling. We describe the application of a new group of diaminoterephthalate-derived fluorescence probes targeting either cysteines in guanylate cyclase-activating proteins, named GCAPs, or azide moieties in covalently attached acyl groups. By monitoring Ca2+-dependent changes in fluorescence emission, we identify Ca2+-sensitive protein regions in GCAPs and correlate conformational changes to protein function.

Key words

Neuronal calcium sensor proteins GCAP Fluorescence Myristoylation Diaminoterephthalates 



This work was supported by a grant from the Deutsche Forschungsgemeinschaft (GRK 1885).


  1. 1.
    Ikura M, Ames JB (2006) Genetic polymorphism and protein conformational plasticity in the calmodulin superfamily: two ways to promote multifunctionality. Proc Natl Acad Sci U S A 103:1159–1164CrossRefGoogle Scholar
  2. 2.
    Philippov PP, Koch K-W (eds) (2006) Neuronal calcium sensor proteins. Nova Publishers, Hauppauge, NYGoogle Scholar
  3. 3.
    Burgoyne RD, Haynes LP (2014) Sense and specificity in neuronal calcium signalling. Biochim Biophys Acta 1853:1921–1932CrossRefGoogle Scholar
  4. 4.
    Ames JB, Ishima R, Tanaka T et al (1997) Molecular mechanics of calcium-myristoyl switches. Nature 389:198–202CrossRefGoogle Scholar
  5. 5.
    Sulmann S, Dell’Orco D, Marino V et al (2014) Conformational changes in calcium-sensor proteins under molecular crowding conditions. Chem Eur J 20:6756–6762CrossRefGoogle Scholar
  6. 6.
    Dizhoor AM, Olshevskaya EV, Henzel WJ et al (1995) Cloning, sequencing, and expression of a 24-kDa Ca2+-binding protein activating photoreceptor guanylyl cyclase. J Biol Chem 270:25200–25206CrossRefGoogle Scholar
  7. 7.
    Howes K, Bronson JD, Dang YL et al (1998) Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2. Invest Ophthalmol Vis Sci 39:867–875PubMedGoogle Scholar
  8. 8.
    Haeseleer F, Sokal I, Li N, Pettenati M et al (1999) Molecular characterization of a third member of the guanylyl cyclase-activating protein subfamily. J Biol Chem 274:6526–6535CrossRefGoogle Scholar
  9. 9.
    Imanishi Y, Yang L, Sokal I et al (2004) Diversity of guanylate cyclase-activating proteins (GCAPS) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes). J Mol Evol 59:2204–2217CrossRefGoogle Scholar
  10. 10.
    Takemoto N, Tachibanaki S, Kawamura S (2009) High cGMP synthetic activity in carp cones. Proc Natl Acad Sci U S A 106:11788–11793CrossRefGoogle Scholar
  11. 11.
    Sulmann S, Vocke F, Scholten A et al (2015) Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding. Sci Rep 5:11228CrossRefGoogle Scholar
  12. 12.
    Hwang JY, Lange C, Helten A et al (2003) Regulatory modes of rod outer segment membrane guanylate cyclase differ in catalytic efficiency and Ca2+-sensitivity. Eur J Biochem 270:3814–3821CrossRefGoogle Scholar
  13. 13.
    Peshenko IV, Olshevskaya EV, Savchenko AB et al (2011) Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors. Biochemistry 50:5590–5600CrossRefGoogle Scholar
  14. 14.
    Koch KW, Dell’Orco D (2013) A calcium relay mechanism in vertebrate phototransduction. ACS Chem Neurosci 4:909–917CrossRefGoogle Scholar
  15. 15.
    Peshenko IV, Dizhoor AM (2004) Guanylyl cyclase-activating proteins (GCAPs) are Ca2+/Mg2+ sensors: implications for photoreceptor guanylyl cyclase (RetGC) regulation in mammalian photoreceptors. J Biol Chem 279:16903–16906CrossRefGoogle Scholar
  16. 16.
    Wu D, Cheung S, Devocelle M et al (2015) Synthesis and assessment of a maleimide functionalized BF2 azadipyrromethene near-infrared fluorochrome. Chem Commun 51:16667–16670CrossRefGoogle Scholar
  17. 17.
    Haimi P, Sikorskaite-Gudziuniene S, Baniulis D (2015) Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment. Proteomics 15:1777–1780CrossRefGoogle Scholar
  18. 18.
    Dietz L, Bosque A, Pankert P et al (2009) Quantitative DY-maleimide-based proteomic 2-DE-labeling strategies using human skin proteins. Proteomics 9:4298–4308CrossRefGoogle Scholar
  19. 19.
    Jewett JC, Bertozzi CR (2010) Cu-free click cycloaddition reactions in chemical biology. Chem Soc Rev 39:1272–1279CrossRefGoogle Scholar
  20. 20.
    Bernard S, Audisio D, Riomet M et al (2017) Bioorthogonal click and release reaction of iminosydnones with cycloalkynes. Angew Chem Int Ed 56:15612–15616 Angew Chem 129: 15818–15822CrossRefGoogle Scholar
  21. 21.
    Mamot A, Sikorski PJ, Warminski M et al (2017) Azido-functionalized 5′ cap analogues for the preparation of translationally active mRNAs suitable for fluorescent labeling in living cells. Angew Chem Int Ed 56:15628–15632 Angew Chem 129: 15834–15838CrossRefGoogle Scholar
  22. 22.
    Tornøe CW, Christensen C, Medal M (2002) Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J Org Chem 67:3057–3064CrossRefGoogle Scholar
  23. 23.
    Rostovtsev VV, Green LG, Fokin VV et al (2002) A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew Chem Int Ed 41:2596–2599 Angew Chem 114: 2708–2711CrossRefGoogle Scholar
  24. 24.
    Amblard F, Cho JH, Schinazi RF (2009) Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition reaction in nucleoside, nucleotide, and oligonucleotide chemistry. Chem Rev 109:4207–4220CrossRefGoogle Scholar
  25. 25.
    Christoffers J (2018) Diaminoterephthalate fluorescence dyes – versatile tools for life sciences and materials science. Eur J Org Chem 2018:2366–2377CrossRefGoogle Scholar
  26. 26.
    Sulmann S, Wallisch M, Scholten A et al (2016) Mapping calcium-sensitive regions in the neuronal calcium sensor GCAP2 by site-specific fluorescence labeling. Biochemistry 55:2567–2577CrossRefGoogle Scholar
  27. 27.
    Freimuth L, Rozzi CA, Lienau C et al (2015) A diaminoterephthalate C60 dyad – a new material for opto-electronic applications. Synthesis 47:1325–1328CrossRefGoogle Scholar
  28. 28.
    Antczak C, Bauvois B, Monneret C et al (2001) A new acivicin prodrug designed for tumor-targeted delivery. Bioorg Med Chem 9:2843–2848CrossRefGoogle Scholar
  29. 29.
    Krapcho PA, Kuell CA (1990) Mono-protected diamines. N-tert-butoxycarbonyl-α,ω-alkanediamines from α,ω-alkanediamines. Synth Commun 20:2559–2564CrossRefGoogle Scholar
  30. 30.
    Hwang JY, Schlesinger R, Koch KW (2001) Calcium-dependent cysteine reactivities in the neuronal calcium sensor guanylate cyclase-activating protein 1. FEBS Lett 508:355–359CrossRefGoogle Scholar
  31. 31.
    Sokal I, Li N, Klug CS, Filipek S et al (2001) Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopy. J Biol Chem 276:43361–43373CrossRefGoogle Scholar
  32. 32.
    Lim S, Peshenko IV, Dizhoor AM et al (2013) Structural insights for activation for retinal guanylate cyclase by GCAP1. PLoS One 8:e81822CrossRefGoogle Scholar
  33. 33.
    Krylov DM, Niemi GA, Dizhoor AM et al (1999) Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases. J Biol Chem 274:10833–10839CrossRefGoogle Scholar
  34. 34.
    Towler DA, Gordon JI, Adams SP et al (1988) The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem 57:69–99CrossRefGoogle Scholar
  35. 35.
    Kollmann H, Becker SF, Shirdel J et al (2012) Probing the Ca2+ switch of the neuronal Ca2+ sensor GCAP2 by time-resolved fluorescence spectroscopy. ACS Chem Biol 7:1006–1014CrossRefGoogle Scholar
  36. 36.
    Robin J, Brauer J, Sulmann S et al (2015) Differential nanosecond protein dynamics in homologous calcium sensors. ACS Chem Biol 10:2344–2352CrossRefGoogle Scholar
  37. 37.
    Tsien R, Pozzan T (1989) Measurement of cytosolic free Ca2+ with Quin2. Methods Enzymol 172:230–262CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biochemistry Group, Department of Neuroscience, Faculty VIUniversity of OldenburgOldenburgGermany
  2. 2.Organic Chemistry, Faculty VInstitut für Chemie, University of OldenburgOldenburgGermany

Personalised recommendations