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.