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Journal Article

Resonance energy transfer in a calcium concentration-dependent cameleon protein

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Subramaniam,  V.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-F26B-8
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.