Resonance energy transfer in a calcium concentration-dependent cameleon protein

Satoshi Habuchi, Mircea Cotlet, Johan Hofkens z, Gunter Dirix, Jan Michiels, Jos Vanderleyden, Vinod Subramaniam, Frans C. De Schryver

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28 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3499-3506
Number of pages8
JournalBiophysical Journal
Volume83
Issue number6
DOIs
StatePublished - Dec 1 2002

ASJC Scopus subject areas

  • Biophysics

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