A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications (original) (raw)

Nature Biotechnology volume 20, pages 87–90 (2002)Cite this article

Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems1. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl−)3. For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl− are desired. Here we describe the development of an improved version of YFP named “Venus”. Venus contains a novel mutation, F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.

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

Access options

Subscribe to this journal

Receive 12 print issues and online access

$209.00 per year

only $17.42 per issue

Buy this article

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

Additional access options:

Similar content being viewed by others

References

  1. Tsien, R.Y. The green fluorescent protein. Ann. Rev. Biochem. 67, 509–544 (1998).
    Article CAS Google Scholar
  2. Crameri, A., Whitehorn, E.A., Tate, E. & Stemmer, W.P.C. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14, 315–319 (1996).
    Article CAS Google Scholar
  3. Jayaraman, S., Haggie, P., Wachter, R.M., Remington, S.J. & Verkman, A.S. Mechanism and cellular applications of a green fluorescent protein–based halide sensor. J. Biol. Chem. 275, 6047–6050 (2000).
    Article CAS Google Scholar
  4. Nagai, T, Sawano, A., Park, E.S. & Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98, 3197–3202 (2001).
    Article CAS Google Scholar
  5. Sawano, A. & Miyawaki, A. Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res. 28, e78 (2000).
    Article CAS Google Scholar
  6. Reid, B.G. & Flynn, G.C. Chromophore formation in green fluorescent protein. Biochemistry 36, 6786–6791 (1997).
    Article CAS Google Scholar
  7. Miyawaki, A. & Tsien, R.Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000).
    Article CAS Google Scholar
  8. Heikal, A.A., Hess, S.T., Baird, G.S., Tsien, R.Y. & Webb, W.W. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).
    Article CAS Google Scholar
  9. Griesbeck, O., Baird, G.S., Campbell, R.E., Zacharias, D.A. & Tsien, R.Y. Reducing the environmental sensitivity of yellow fluorescent protein. J. Biol. Chem. 276, 29188–29194 (2001).
    Article CAS Google Scholar
  10. Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R.Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc. Natl. Acad. Sci. USA 96, 2135–2140 (1999).
    Article CAS Google Scholar
  11. Lang, T. et al. Ca2+-triggered peptide secretion in single cells imaged with green fluorescent protein and evanescent-wave microscopy. Neuron 18, 857–863 (1997).
    Article CAS Google Scholar
  12. Tsuboi, T., Zhao, C., Terakawa, S. & Rutter, G.A. Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event. Curr. Biol. 10, 1307–1310 (2000).
    Article CAS Google Scholar
  13. Pouli, A., Kennedy, H., George Schofield, J. & Rutter, G. Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase. Biochem. J. 331, 669–675 (1998).
    Article CAS Google Scholar
  14. Angleson, J.K. & Betz, W.J. Monitoring secretion in real time: capacitance, amperometry and fluorescence compared. Trends Neurosci. 20, 281–187(1997).
    Article CAS Google Scholar
  15. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).
    Article CAS Google Scholar
  16. Cadwell, R.C. & Joyce, G.F. Mutagenic PCR. PCR Methods Appl. 3, S136–S140 (1994).
    Article CAS Google Scholar

Download references

Acknowledgements

The plasmid construct pEGFP-N1-NPY was provided by W. Almers. We are grateful to W. Almers and R.Y. Tsien for valuable comments, M. Yamamoto-Hino, Y. Kawano, K. Shimizu, T. Miyata, and K. Nakamura for technical advice, and H. Kuramochi for technical assistance. This work was partly supported by grants from CREST (the Japan Science and Technology Corporation) to A.M., the Japanese Ministry of Education, Science and Culture to A.M., Special Postdoctoral Researcher Program of RIKEN to T.N., and President's Special Research Grant of RIKEN to T.N.

Author information

Authors and Affiliations

  1. Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, 351-0198, Saitama, Japan
    Takeharu Nagai, Eun Sun Park & Atsushi Miyawaki
  2. Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, 351-0198, Saitama, Japan
    Keiji Ibata & Katsuhiko Mikoshiba
  3. Laboratory for Molecular Dynamics of Mental Disorder, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, 351-0198, Saitama, Japan
    Mie Kubota

Authors

  1. Takeharu Nagai
    You can also search for this author inPubMed Google Scholar
  2. Keiji Ibata
    You can also search for this author inPubMed Google Scholar
  3. Eun Sun Park
    You can also search for this author inPubMed Google Scholar
  4. Mie Kubota
    You can also search for this author inPubMed Google Scholar
  5. Katsuhiko Mikoshiba
    You can also search for this author inPubMed Google Scholar
  6. Atsushi Miyawaki
    You can also search for this author inPubMed Google Scholar

Corresponding author

Correspondence toAtsushi Miyawaki.

Rights and permissions

About this article

Cite this article

Nagai, T., Ibata, K., Park, E. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.Nat Biotechnol 20, 87–90 (2002). https://doi.org/10.1038/nbt0102-87

Download citation

This article is cited by

Associated content