A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy


Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions1,2,3,4,5. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.

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Figure 1: In vitro evolution of a chromoprotein from the stony coral Montipora sp. into a fluorescent protein with a large Stokes shift.
Figure 2: Single laser wavelength (458 nm) excitation FCCS using mKeima and CFP to monitor proteolysis by caspase-3.
Figure 3: Single laser wavelength (458 nm) excitation FCCS using mKeima and CFP to monitor the Ca2+-dependent association between CaM and CaMKI.
Figure 4: Simultaneous six-color imaging of subcellular structures in a Vero cell using a single laser line (458 nm).

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The authors would like to thank K. Iwao and S. Hosaka at the Akajima Marine Science Laboratory for acquiring the stony coral animals, Y. Isogai for assistance with analytical centrifugation, F. Ishidate, K. Weisshart, B. Zimmerman, Y. Hasegawa for assistance with FCCS measurements and spectral imaging, and K. Ishihara, H. Watanabe, T. Fukano, and M. Hirano for assistance with multi-color imaging and fluorescence lifetime measurements. This work was partly supported by grants from Japan MEXT Grant-in-Aid for Scientific Research on priority areas, NEDO (the New Energy and Industrial Technology Development Organization), HFSP (the Human Frontier Science Program), and RIKEN Strategic Research Program.

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Correspondence to Atsushi Miyawaki.

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Competing interests

S.K. and T.A. get salaries from Amalgaam and Medical Biological Laboratories, which will sell the products presented in this paper.

Supplementary information

Supplementary Fig. 1

An amino-acid sequence alignment of DsRed with #20, #20-9115, tKeima, dKeima, mKeima, and dKeima570. (PDF 194 kb)

Supplementary Fig. 2

Equilibrium radial absorbance profiles obtained by analytical ultracentrifugation for Keima variants. (PDF 288 kb)

Supplementary Fig. 3

Quantitative performance of FCCS using an mKeima-CFP pair. (PDF 262 kb)

Supplementary Fig. 4

Detection of the Ca2+-dependent association between CaM and CaMKI with the SL-FCCS technique using mKeima and CFP. (PDF 219 kb)

Supplementary Fig. 5

Single laser wavelength (458 nm) excitation FCCS to monitor the Ca2+-dependent association between CaM and M13 in a living HeLa cell, which was expressing mCFP-CaM and M13-mKeima. (PDF 251 kb)

Supplementary Fig. 6

Simultaneous imaging of [Ca2+]c and mitochondrial morphology. (PDF 937 kb)

Supplementary Fig. 7

Excitation (broken lines) and emission (solid lines) spectra of dKeima and dKeima570. (PDF 215 kb)

Supplementary Table 1

Spectral characteristics of CFP, Keima, dKeima, mKeima, and dKeima570. (PDF 85 kb)

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Kogure, T., Karasawa, S., Araki, T. et al. A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat Biotechnol 24, 577–581 (2006). https://doi.org/10.1038/nbt1207

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