Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging.
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.
Villoing
Aude
A
Laboratoire de Chimie Physique, UMR 8000, Université Paris-Sud 11 and CNRS, Orsay 91405, France.
Ridhoir
Myriam
M
Cinquin
Bertrand
B
Erard
Marie
M
Alvarez
Luis
L
Vallverdu
Germain
G
Pernot
Pascal
P
Grailhe
Régis
R
Mérola
Fabienne
F
Pasquier
Hélène
H
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
C485182:Cyan Fluorescent Protein; D050505:Mutant Proteins; D049452:Green Fluorescent Proteins