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. 2013 Apr 3;4(4):299–309. doi: 10.1007/s13238-013-2103-4

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Yingxiao Chen 1, Xianqiang Song 1, Sheng Ye 1, Lin Miao 1, Yun Zhu 1, Rong-Guang Zhang 1,✉, Guangju Ji 1,✉
PMCID: PMC4875517  PMID: 23549615

Abstract

Genetically encoded Ca2+ indicators (GECI) are important for the measurement of Ca2+in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca2+ binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca2+ signals in intact organs to facilitate the further improvement of GCaMP sensors.

Keywords: genetically encoded calcium indicator, mutants, crystal structure, fluorescentintensity, dimerization

Footnotes

These authors contributed equally to the work.

Contributor Information

Rong-Guang Zhang, Email: rzhang@sun5.ibp.ac.cn.

Guangju Ji, Email: gj28@ibp.ac.cn.

References

  1. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral GJ, Grosse-Kunstleve RW, et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–221. doi: 10.1107/S0907444909052925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akerboom J, Rivera JD, Guilbe MM, Malave EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER. Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J Biol Chem. 2009;284:6455–6464. doi: 10.1074/jbc.M807657200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borghuis BG, Tian L, Xu Y, Nikonov SS, Vardi N, Zemelman BV, Looger LL. Imaging light responses of targeted neuron populations in the rodent retina. J Neurosci. 2011;31:2855–2867. doi: 10.1523/JNEUROSCI.6064-10.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci U S A. 1997;94:2306–2311. doi: 10.1073/pnas.94.6.2306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chattoraj M, King BA, Bublitz GU, Boxer SG. Ultra fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci U S A. 1996;93:8362–8367. doi: 10.1073/pnas.93.16.8362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci. 2010;13:1433–1440. doi: 10.1038/nn.2648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Elsliger MA, Wachter RM, Hanson GT, Kallio K, Remington SJ. Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry. 1999;38:5296–5301. doi: 10.1021/bi9902182. [DOI] [PubMed] [Google Scholar]
  8. Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
  9. Hires SA, Tian L, Looger LL. Reporting neural activity with genetically encoded calcium indicators. Brain Cell Biol. 2008;36:69–86. doi: 10.1007/s11068-008-9029-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ji G, Feldman ME, Deng KY, Greene KS, Wilson J, Lee JC, Johnston RC, Rishniw M, Tallini Y, Zhang J, et al. Ca2+-sensing transgenic mice: postsynaptic signaling in smooth muscle. J Biol Chem. 2004;279:21461–21468. doi: 10.1074/jbc.M401084200. [DOI] [PubMed] [Google Scholar]
  11. Jung G, Wiehler J, Zumbusch A. The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222. Biophys J. 2005;88:1932–1947. doi: 10.1529/biophysj.104.044412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kotlikoff MI. Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J Physiol. 2007;578:55–67. doi: 10.1113/jphysiol.2006.120212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kummer AD, Wiehler J, Rehaber H, Kompa C, Steipe B, Michel-Beyerle ME. Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the green fluorescent protein (GFP) J Physical Chem B. 2000;104:4791–4798. doi: 10.1021/jp9942522. [DOI] [Google Scholar]
  14. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. Phaser crystallographic software. J Appl Crystallogr. 2007;40:658–674. doi: 10.1107/S0021889807021206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Murshudov GN, Vagin AA, Dodson EJ. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
  16. Nagai T, Sawano A, Park ES, Miyawaki A. Circularly permuted green fluorescent proteins engineered to sense Ca2+ Proc Natl Acad Sci U S A. 2001;98:3197–3202. doi: 10.1073/pnas.051636098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ohkura M, Matsuzaki M, Kasai H, Imoto K, Nakai J. Genetically encoded bright Ca2+ probe applicable for dynamic Ca2+ imaging of dendritic spines. Anal Chem. 2005;77:5861–5869. doi: 10.1021/ac0506837. [DOI] [PubMed] [Google Scholar]
  18. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. Crystal structure of the Aequorea victoria green fluorescent protein. Science. 1996;273:1392–1395. doi: 10.1126/science.273.5280.1392. [DOI] [PubMed] [Google Scholar]
  19. Seelig JD, Chiappe ME, Lott GK, Dutta A, Osborne JE, Reiser MB, Jayaraman V. Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior. Nat Methods. 2010;7:535–540. doi: 10.1038/nmeth.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shiba Y, Fernandes S, Zhu WZ, Filice D, Muskheli V, Kim J, Palpant NJ, Gantz J, Moyes KW, Reinecke H, et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature. 2012;489:322–325. doi: 10.1038/nature11317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Stoner-Ma D, Jaye AA, Matousek P, Towrie M, Meech SR, Tonge PJ. Observation of excited-state proton transfer in green fluorescent protein using ultrafast vibrational spectroscopy. J Am Chem Soc. 2005;127:2864–2865. doi: 10.1021/ja042466d. [DOI] [PubMed] [Google Scholar]
  22. Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, et al. Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ in-dicator GCaMP2. Proc Natl Acad Sci U S A. 2006;103:4753–4758. doi: 10.1073/pnas.0509378103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tian L, Hires SA, Mao T, Huber D, Chiappe ME, Chalasani SH, Petreanu L, Akerboom J, McKinney SA, Schreiter ER, et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat Methods. 2009;6:875–881. doi: 10.1038/nmeth.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. [DOI] [PubMed] [Google Scholar]
  25. Wang Q, Shui B, Kotlikoff MI, Sondermann H. Struc tural basis for calcium sensing by GCaMP2. Structure. 2008;16:1817–1827. doi: 10.1016/j.str.2008.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yang F, Moss LG, Phillips GN., Jr The molecular structure of green fluorescent protein. Nat Biotechnol. 1996;14:1246–1251. doi: 10.1038/nbt1096-1246. [DOI] [PubMed] [Google Scholar]
  27. Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, et al. An expanded palette of genetically encoded Ca(2)(+) indicators. Science. 2011;333:1888–1891. doi: 10.1126/science.1208592. [DOI] [PMC free article] [PubMed] [Google Scholar]

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