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  Resonance energy transfer in a calcium concentration-dependent cameleon protein

Habuchi, S., Cotlet, M., Hofkens, J., Dirix, G., Michiels, J., Vanderleyden, J., et al. (2002). Resonance energy transfer in a calcium concentration-dependent cameleon protein. Biophysical Journal, 83(6), 3499-3506. Retrieved from http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B94RW-4V466WB-1V-1&_cdi=56421&_user=38661&_pii=S0006349502753496&_orig=search&_coverDate=12%2F31%2F2002&_sk=999169993&view=c&wchp=dGLbVlW-zSkWA&md5=7689621a4a2332505e2382f52eee0115&ie=/sdarticle.pdf.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0012-F26B-8 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EC7C-E
Genre: Journal Article

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599419.pdf (Publisher version), 258KB
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Habuchi, S., Author
Cotlet, M., Author
Hofkens, J., Author
Dirix, G., Author
Michiels, J., Author
Vanderleyden, J., Author
Subramaniam, V.1, Author              
De Schryver, F. C., Author
Affiliations:
1Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society, ou_578628              

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 Abstract: We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca2+ called cameleons using steady- state and time-resolved spectroscopy of the full construct and of the component green fluorescent protein mutants, namely ECFP (donor) and EYFP (acceptor). EYFP displays a complicated photophysical behavior including protonated and deprotonated species involved in an excited-state proton transfer. When EYFP is excited in the absorption band of the protonated species, a fast nonradiative deactivation occurs involving almost 97% of the excited protonated population and leading to a low efficiency of excited-state proton transfer to the deprotonated species. ECFP displays a multiexponential fluorescence decay with a major contributing component of 3.2 ns. The time- resolved fluorescence data obtained upon excitation at 420 nm of Ca2+-free and Ca2+-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca2+ binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca2+ binding, the time-resolved data demonstrate the existence of three distinct conformations/populations within the investigated sample. Although the mechanism of the interconversion between the different conformations and the extent of interconversion are still unclear, the time-resolved fluorescence data offer an estimation of the rate constants, of the efficiency of the energy transfer, and of the donor- acceptor distances in the Ca2+-free and Ca2+-bound YC3.1 samples.

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Language(s): eng - English
 Dates: 2004-07-302002-12
 Publication Status: Published in print
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 Rev. Method: Peer
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 83 (6) Sequence Number: - Start / End Page: 3499 - 3506 Identifier: -