Synthesis and Application of Fluorescence-Labeled Ras-Proteins for Live-Cell Imaging

摘要

Live-cell imaging employing fluorescent-labeled probes is rapidly developing into one of the most important techniques of biological research. A significant bottleneck in the application of this technology is the efficient and site-selective labeling of proteins. Molecular biological introduction of green fluorescent protein (GFP) and congeners like cyan fluorescent protein (CFP) has emerged as a powerful tool for this purpose. However, since the biological fluorophore GFP has a mass of 27 kDa, its influence on the in-vivo readout cannot be excluded, in particular if protein-protein interactions are involved. For instance, restrictions in the use of the GFP marker due to steric constraints and undesired interactions were reported in studies of tubule formation and of the secretory pathway in yeast. An additional limitation is given by the overlapping emission and excitation spectra of the accessible fluorescent proteins that restrict utilization in FRET (fluorescence resonance energy transfer) assays or dual-wavelength confocal microscopy. Also, GFP shows flickering where phases of active and inactive fluorescence emission alternate. These drawbacks can be overcome by the use of semisynthetic proteins carrying small fluorophores, and this general approach has been widely explored.