Chromophore Transformations in Red Fluorescent Proteins

Abstract

The discovery of Anthozoa homologs of the green fluorescent protein (GFP) from jellyfish Aequorea victoria, which emit not only green but also yellow, orange, and red fluorescence, provided a powerful boost for in vivo labeling due to the colors and biochemical features never before encountered in GFP variants.1,2 GFP and several Anthozoa fluorescent proteins (FPs) have been developed into monomers suitable for protein tagging, such as: (i) permanently fluorescent conventional blue FP, yellow FPs, orange FPs, red FPs (RFPs), (ii) permanently fluorescent GFPs and RFPs with a large Stokes shift fluorescence emission (LSS-FPs), (iii) irreversibly photoactivatable/photoswitchable GFPs and RFPs,3,4 and (iv) fluorescent timers (FTs).5,6 Among various fluorescent probes, the most valuable for deep-tissue and whole-body imaging are the red-shifted FPs because of reduced autofluorescence, low light-scattering, and minimal absorbance at longer imaging wavelengths. The mechanisms of formation of the GFP-like chromophore and its transformations were studied and described very well, however only a few reviews of those for RFPs are available 2,7. Several mechanisms of the autocatalytic and photoinduced formation of the red chromophores have been proposed.7 Because of the complexity of the red chromophore transformations, no general integrating scheme has been suggested. Despite the numerous data available, there are long lasting contradictions about the formation of the red chromophore. From the discovery of a DsRed FP, the formation of a DsRed-like chromophore was commonly suggested to occur through a green GFP-like intermediate form,8 and only several recent publications uncovered that the formation of the DsRed-like chromophore occurs via a TagBFP-like blue intermediate form, not via the GFP-like one. More than 140 crystal structures are currently available for FPs of different classes, and some of them are for the same FP in a different state or containing different mutations. An overview of the structural data together with FP spectral and photochemical properties illustrate the relationship between the FPs’ structure and function. Here, we focus on a description of the chromophores in RFPs, suggest the mechanisms of the red chromophores formation and its further modifications, and attempt to discover general postulates for this complex chemistry. We also provide insights into how the red chromophore chemistry and the RFP crystal structures are translated into RFPs function. Lastly, we descuss major applications of RFPs in the modern imaging techniques.