Improving autofluorescent proteins: Comparative studies of the effective brightness of Green Fluorescent Protein (GFP) mutants

Abstract

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 Phi(Fl) (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 photostationary conditions. The determination of the excitable fraction [B] and its fluorescence quantum yield Phi(Fl) 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 YFP-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.

Fluorescent proteins for single‐molecule fluorescence applications

“…Since the discovery and isolation of green fluorescent protein (GFP), fluorescent fusion proteins have played an ever more important role in fluorescence microscopy and spectroscopy [1,2]. The ease of expressing translationally fused fluorescent proteins as tags of particular target proteins allows chemical labeling to be circumvented, which is in particular important for noninvasive in-vivo fluorescence microscopy [3,4].…”

Photostability of green and yellow fluorescent proteins with fluorinated chromophores, investigated by fluorescence correlation spectroscopy

“…These values are only slightly higher than the reported values for EGFP and EYFP, measured by single-molecule fluorescence microscopy [11]. Note that the fluorescence quantum yield for EYFP is higher than for EGFP making EYFP even more favourable for fluorescence applications [19]. In addition, the photon detection probability is not uniform across the (inhomogeneous) excitation profile.…”

“…C is of importance in discussing photostability as it can be related to the fraction of molecules in a dark state, [D], according to eq. 2 [19].…”

“…Not all proteins of one mutant are in a bright state but undergo isomerization into dark states. As in a previous study, we refer σ bl to the bright fraction B which can be calculated by the contrast C [19] (fig. 4b).…”

Efficient Photoconversion Distorts the Fluorescence Lifetime of GFP in Confocal Microscopy: A Model Kinetic Study on Mutant Thr203Val

“…[30] While selective PC of the acceptor in a FRET pair would falsely indicate a separation of donor and acceptor, the reverse is true for donor PC, even in microscopy with cw excitation.…”