Engineering red fluorescent proteins

Fluorescent proteins (FPs) are used as genetic labels to study processes in live cells by using fluorescence microscopy. Multiple processes can be observed simultaneously, when FPs in multiple colors are used. Currently, red fluorescent proteins (RFPs) are not optimal for quantitative cellular imaging. The aim of the research presented in this thesis is to create an improved monomeric red fluorescent protein (mRFP). Initially, we applied established methods to the development of mRFPs. However, we found that the development of mRFPs was more complex and screening on merely one parameter is not sufficient. Therefore, we developed a multi-parameter screening method (Chapter 1). The development of this multi-parameter screening method coincided with the development of improved mRFPs and led to the mScarlet family (Chapter 2). mScarlet is the brightest mRFP with a record quantum yield in its spectral class. Two other important variants are developed: mScarlet-I which shows enhanced maturation and mScarlet-H which displays extreme photostability. We quantitively compared the new mScarlets to existing RFPs in vitro and in vivo. Moreover, photophysical properties of the mScarlet family and several well-known RFPs are studied (Chapter 3). Many photophysical properties critically depend on both pH and illumination and are different for each RFP variant. This makes it a major challenge to optimize and screen for RFPs with a particular combination of favorable photophysical properties. The developed methods and new insights will assist future development of FPs of any color. Ultimately, mScarlet itself can serve as a new template for mRFP optimization

Multiparameter screening method for developing optimized red-fluorescent proteins

Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using mutagenesis. The optimization process through screening is often based solely on bacterial colony brightness, but multiple parameters ultimately determine the performance of an optimal FP. Instead of characterizing other properties after selection, we developed a multiparameter screening method based on four critical parametersscreened simultaneously: fluorescence lifetime, cellular brightness, maturation efficiency, and photostability. First, a high-throughput primary screen (based on fluorescence lifetime and cellular brightness using a mutated FP library) is performed in bacterial colonies. A secondary multiparameter screen based on all four parameters, using a novel bacterial-mammalian dual-expression vector enables expression of the best FP variants in mammalian cell lines. A newly developed automated multiparameter acquisition and cell-based analysis approach for 96-well plates further increased workflow efficiency. We used this protocol to yield the record-bright mScarlet, a fast-maturating mScarlet-I, and a photostable mScarlet-H. This protocol can also be applied to other FP classes or Förster resonance energy transfer (FRET)-based biosensors with minor adaptations. With an available mutant library of a template FP and a complete and tested laboratory setup, a single round of multiparameter screening (including the primary bacterial screen, secondary mammalian cell screen, sequencing, and data processing) can be performed within 2 weeks

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor

Quantitative Determination of Dark Chromophore Population Explains the Apparent Low Quantum Yield of Red Fluorescent Proteins

The fluorescence quantum yield of four representative red fluorescent proteins mCherry, mKate2, mRuby2, and the recently introduced mScarlet was investigated. The excited state lifetimes were measured as a function of the distance to a gold mirror in order to control the local density of optical states (LDOS). By analyzing the total emission rates as a function of the LDOS, we obtain separately the emission rate and the nonradiative rate of the bright states. We thus obtain for the first time the bright state quantum yield of the proteins without interference from dark, nonemitting states. The bright state quantum yields are considerably higher than previously reported quantum yields that average over both bright and dark states. We determine that mCherry, mKate2, and mRuby2 have a considerable fraction of dark chromophores up to 45%, which explains both the low measured quantum yields of red emitting proteins reported in the literature and the difficulties in developing high quantum yield variants of such proteins. For the recently developed bright mScarlet, we find a much smaller dark fraction of 14%, accompanied by a very high quantum yield of the bright state of 81%. The presence of a considerable fraction of dark chromophores has implications for numerous applications of fluorescent proteins, ranging from quantitative fluorescence microscopy to FRET studies to monitoring protein expression levels. We recommend that future optimization of red fluorescent proteins should pay more attention to minimizing the fraction of dark proteins