title: Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging.
abstract: We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.
authors.affiliation: Laboratoire de Chimie Physique, UMR 8000, Université Paris-Sud 11 and CNRS, Orsay 91405, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Villoing
authors.forename: Myriam
authors.initials: M
authors.lastname: Ridhoir
authors.forename: Bertrand
authors.initials: B
authors.lastname: Cinquin
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
authors.forename: Luis
authors.initials: L
authors.lastname: Alvarez
authors.forename: Germain
authors.initials: G
authors.lastname: Vallverdu
authors.forename: Pascal
authors.initials: P
authors.lastname: Pernot
authors.forename: Régis
authors.initials: R
authors.lastname: Grailhe
authors.forename: Fabienne
authors.initials: F
authors.lastname: Mérola
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
mesh_terms: D000818:Animals; D002460:Cell Line; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
chemical_list: C485182:Cyan Fluorescent Protein; D050505:Mutant Proteins; D049452:Green Fluorescent Proteins