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.

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

“…It is to be noted, however, that the excitation wavelength was at 430 nm instead at 400 nm and that the buffer composition was also different as compared to the previous experiment with ECFP. This resolution in three lifetime components instead of two using the MEM approach has been observed before and illustrates again that the ECFP fluorescence decay is heterogeneous and the distributed lifetimes are sensitive to external factors (Villoing et al 2008). MEM analysis of the donor fluorescence decay in the FRET Table 2 construct recovered four distributed lifetimes, where the short lifetimes (0.12 and 0.49 ns) must be ascribed to FRET, since they do not show up in the lifetime distribution of ECFP alone.…”

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression

“…The relevant P values are reported in the figure panels and legends. References (57)(58)(59)(60)(61)(62)(63)(64)(65) …”

TRANSPARENT TESTA GLABRA1 and GLABRA1 Compete for Binding to GLABRA3 in Arabidopsis

“…Therefore, a model to fit the data with a minimal number of parameters has to be applied in conjunction with a maximum likelihood estimator (Schaffer et al, 1999;Eggeling et al, 2001;Widengren et al, 2006;Weidtkamp-Peters et al, 2009;Sisamakis et al, 2010). We are aware that CFP alone already has a multiexponential fluorescence decay (Villoing et al, 2008), which becomes even more complex in the presence of additional FRET species. This generally multiexponential decay is approximated in the subsequent fluorescence lifetime analysis by a fluorescenceweighted average lifetime, t. Therefore, we used a monoexponential model function with two variables (fluorescence lifetime t and scatter contribution g), as described elsewhere (Stahl et al, 2013), fitted with a maximum likelihood estimator.…”