title: Resonance energy transfer in a calcium concentration-dependent cameleon protein.
abstract: We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca(2+) 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 Ca(2+)-free and Ca(2+)-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca(2+) binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca(2+) 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 Ca(2+)-free and Ca(2+)-bound YC3.1 samples.
authors.affiliation: Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Habuchi
authors.forename: Mircea
authors.initials: M
authors.lastname: Cotlet
authors.forename: Johan
authors.initials: J
authors.lastname: Hofkens
authors.forename: Gunter
authors.initials: G
authors.lastname: Dirix
authors.forename: Jan
authors.initials: J
authors.lastname: Michiels
authors.forename: Jos
authors.initials: J
authors.lastname: Vanderleyden
authors.forename: Vinod
authors.initials: V
authors.lastname: Subramaniam
authors.forename: Frans C
authors.initials: FC
authors.lastname: De Schryver
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000528:radiation effects; D002118:Calcium / Q000032:analysis / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000528:radiation effects; D004735:Energy Transfer / Q000528:radiation effects*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D002147:Calmodulin; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D002118:Calcium