Improving autofluorescent proteins: Comparative studies of the effective brightness of green fluorescent protein (GFP) mutants

We study the photophysical behavior of 8 mutants of Green Fluorescent Protein (GFP) using fluorescence correlation spectroscopy (FCS) on the single molecule level and double resonance excitation of bulk samples. Experimental data reported here and the previously published data on the RH/R- equilibrium and fluorescence quantum yields (1)Phi(FI) (Jung et al., 2005; Biophys J 88:1932-1947) are analyzed with respect to single molecule as well as conventional fluorescence microscopy. The fraction of GFP molecules in a dark state, [D], reduces the effective absorption cross section under pbotostationary conditions. The determination of the excitable fraction [B] and its fluorescence quantum yield Phi(FI) gives the effective brightness Phi(eff). Our results show that in its wavelength range, eGFP is, among the GFPs, the best fluorophore for most microscopic applications. However, in the red shifted Y-FP-proteins, there is still potential for improvement, since a pronounced dark state population is detectable in all mutants investigated so far. We propose to use the mutant T203Y/E222Q in imaging studies, whenever the expression yield is not a limiting factor. In FCS experiments, where the useful concentration range of the expressed molecules is restricted to concentrations below micromolarity, our data suggest the use of wt-GFP or mutant T203Y, as these represent photochemical buffers. Both mutants might surpass the limitations given by out-of-focus bleaching in live cell microscopy.