Molecular basis of photochromism of a fluorescent protein revealed by direct 13C detection under laser illumination
Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of 13C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between Cα and Cβ of Y63 in the chromophore, the β-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of β-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.
KeywordsFluorescent protein Photochromism Polymorphism In magnet laser illumination 13C direct-detection
We acknowledge Dr. R. Kato for valuable advice. We thank Dr. C. Marshall for careful reading of the manuscript, and Dr. R. Ando, Ms. K.I. Tong, Ms. K. Otsuki, and Mr. M. Usui for technical assistance. This work was partly supported by grants from the Human Frontier Science Program, Molecular Ensemble Program at RIKEN, Japan MEXT Grant-in-Aid for Scientific Research on priority areas, Japan MEXT and Japan Society for the Promotion of Science for Grants-in-Aid for Scientific Research B, and Canadian Institutes for Health Research.
- Bradbury JH, Ramesh V (1985) 1H n.m.r. studies of insulin. Assignment of resonances and properties of tyrosine residues. Biochem J 229:731–737Google Scholar
- Dedecker P, Hotta J, Flors C, Sliwa M, Uji-I H, Roeffaers MB, Ando R, Mizuno H, Miyawaki A, Hofkens J (2007) Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J Am Chem Soc 129:16132–16141CrossRefGoogle Scholar
- Egan W, Shindo H, Cohen JS (1978) On the tyrosine residues of ribonuclease A. J Biol Chem 253:16–17Google Scholar
- Kaptein R (1982) In: Berliner LJ, Reuben J (eds) Biological magnetic resonance, vol 4. Plenum Press, New York, pp 145–191Google Scholar
- Kwon OY, Kwon IC, Song HK, Jeon H (2008) Real-time imaging of NF-AT nucleocytoplasmic shuttling with a photoswitchable fluorescence protein in live cells. Biochim Biophys Acta 1780:1403–1407Google Scholar
- Scheffler JE, Cottrell CE, Berliner LJ (1985) An inexpensive, versatile sample illuminator for photo-CIDNP on any NMR spectrometer. J Magn Reson 63:199–201Google Scholar
- Wilbur DJ, Allerhand A (1976) Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo. J Biol Chem 251:5187–5194Google Scholar