Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system

Abstract

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37 °C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

“…S17). Even though a single protein was synthesized, we observed different rates of appearance for Y-FAST and mCherry fluorescence: Although Y-FAST could already be detected as soon as 10 min after the initiation of the protein synthesis, reaching saturation within 90 min, the mCherry signal only started to appear after 50 min and took over 4 h to reach saturation as a result of the slow maturation of its chromophore (32,33). In addition, we compared the expression of mCherry−Y-FAST with that of EGFP and Venus (34), reported to mature within 10 min and 40 min in vitro, respectively (35), the bilirubin-inducible UnaG, and the Firefly luciferase, the latter often used as a reporter of protein synthesis.…”

Ratiometric Imaging of pH Probes

“…Our ability to measure ion fluxes in real time was revolutionized in the 1980s by the development of high-affinity fluorescent Ca 2+ dyes (Grynkiewicz, Poenie, & Tsien, 1985;Macdonald, Chen, & Mueller, 2012) and H + dyes (Paradiso, Tsien, & Machen, 1984;Shaner et al, 2004). The pH-sensitive polar fluorescein derivative 2 0 ,7 0 -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF) remains the most widely used pHi indicator.…”

“…23.2B) may be aggregates of misfolded protein or represent protein in endosomes or lysosomes where a lower pH would quench pHluorin fluorescence. For the mCherry-pHluorin fusion protein, maturation rates of mCherry ($155 min) are slower than pHluorin (GFP, $15 min) (Macdonald et al, 2012). Therefore, temporally regulated expression constructs may show increased ratio as a consequence of immature mCherry.…”

“…Where appropriate, we often draw a circle and copy and paste to select multiple ROI. Alternately, tracing individual cell features (such as focal adhesions; Choi et al, 2013;Macdonald et al, 2012) may need to be accomplished by hand. When measuring individual cells, we typically select 20-30 cells using individual ROIs, while for tissue measurements, we select 3-5 ROIs per piece of tissue.…”

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

“…The variable extent of maturation complicates analysis of protein stoichiometry and colocalization assays of protein complexes in SiMPull. The long, oxygendependent maturation times of many fluorescent proteins (Khmelinskii et al 2012;Macdonald et al 2012) also preclude their use in studying transiently expressed proteins or expression under anaerobic conditions since the FP may fail to mature and remain "dark" in the SiMPull assay. Finally, the photostability and brightness of the proteins themselves can also limit functional assays since blinking or rapid photobleaching of the FP (Dickson et al 1997;Kubitscheck et al 2000;Hendrix et al 2008) may complicate analysis of complexes isolated by SiMPull.…”