Directed molecular evolution to design advanced red fluorescent proteins

Abstract

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Figure 1

Steps in the directed molecular evolution of fluorescent probes. Vertical arrows indicate the typical order of steps. Horizontal arrows represent possible transitions between the steps of molecular evolution, which can be repeated several times in different order.

Figure 2

Major chemical transformations of the chromophores in red fluorescent proteins. (a–c) Transformations in fluorescent protein subfamilies derived from red fluorescent protein (a), mCherry (b) and TagRFP (c). The colored shading of the chemical structures (a) and chromophore numbers (b,c) correspond to the spectral range of the chromophore fluorescence emission. Gray shading denotes the nonfluorescent state; [H] denotes reduction; and [O] denotes oxidation. The chromo states (structures 5, 10 and 13) are not necessarily caused by a cis-trans chromophore isomerization but may result from modifications of the chromophore environment of the same isoform that decrease quantum yield. hv, photon.

Figure 3

Methods that could improve molecular evolution of fluorescent proteins. (a–e) Schematics depict eukaryotic cell–based mutagenesis methods (a,b) and advanced protein expression systems (c–e). Cylinders denote fluorescent protein molecules. Error-prone replication of virus (a) causes point mutations in the viral genome containing a target fluorescent protein gene; after several rounds of replication, the cell expresses mutated fluorescent protein genes. Somatic hypermutations and gene conversion in eukaryotic cells (b) allow for creation of large random mutant gene libraries during cell proliferation (note that only one type of fluorescent protein mutant is produced per cell). Expression of fluorescent protein libraries (c) in thermophilic bacteria for selection of more stable fluorescent proteins. Surface display (d) of fluorescent protein libraries could facilitate screening for fluorescent protein stability under different environmental conditions or for fluorescent protein–based biosensors. In vitro compartmentalization (e) of bacteria in water-oil-water or water-agarose-water droplets should enable screening for fluorescent protein–based biosensors.

Figure 4

Possible FACS-based screening approaches for red-shifted fluorescent proteins. The respective red fluorescent proteins expected to result from each method are listed on the right. The schematic depicts cells or other hosts expressing fluorescent proteins being mixed with ligand, substrate or metabolite with different delays before fluorescence screening. A standard one-photon laser excites flowing cells, and the resultant fluorescent signal is dispersed with a diffraction grating (triangle) and projected onto an array detector (rectangle) for recording of a complete emission spectrum. A two-photon laser excites the cells with two low-energy photons (hv₁) and the resulting fluorescence emission (hv₂) is detected. Linearly polarized laser excitation and the emitted fluorescence signals have different degrees of polarization depending on the size of fluorescent molecules and FRET efficiency between them. Cylinders denote fluorescent proteins in monomeric and dimeric states. Modulated excitation (yellow sinusoid) results in a phase shift, Δϕ, between the fluorescence emission (red) and side-scattered excitation light (SSC; yellow), which is used to compute the average fluorescence lifetime of fluorescent proteins in a cell.