From EosFP to mIrisFP: structure‐based development of advanced photoactivatable marker proteins of the GFP‐family

Abstract

Fluorescent proteins from the GFP family have become indispensable imaging tools in life sciences research. In recent years, a wide variety of these proteins were discovered in non-bioluminescent anthozoa. Some of them feature exciting new properties, including the possibility to change their fluorescence quantum yield and/or color by irradiating with light of specific wavelengths. These photoactivatable fluorescent proteins enable many interesting applications including pulse-chase experiments and super-resolution imaging. In this review, we discuss the development of advanced variants, using a structure-function based, molecular biophysics approach, of the photoactivatable fluorescent protein EosFP, which can be photoconverted from green to red fluorescence by ~400 nm light. A variety of applications are presented that demonstrate the versatility of these marker proteins in live-cell imaging.

A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications

“…Light-driven switching of RSFPs generally involves an isomerization of the chromophore, frequently coupled with a change of its protonation state (18,28,29). Similar to Dronpa and its derivatives, mGeos show two absorption peaks: a major peak at approximately 502 nm and a minor peak at 388 nm ( Fig.…”

“…The existence of the two forms suggests that light might induce a structural rearrangement with the different protonation states of the chromophore. Based on the structures of the mEos2 homologs EosFP (15) and IrisFP (29), X in XYG of mGeos is located at the far end of the chromophore away from Tyr-63, but near Glu-212 and Gln-38, which play pivotal roles in stabilizing the planar structure and π-system of the chromophore by hydrogen-bonds through a water molecule. We speculate that different X in XYG may interact with different amino acids near the chromophore, and indirectly influence the protonation equilibria of the Tyr-63 by changing electrostatic surface potentials and the large π-system.…”

Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants

“…Mutant and wildtype desmin were fused either with a thermostable variant of the green to red convertible fluorescent protein mEosFP termed mEosFPthermo (31)(32)(33) or with the green photoswitchable mIrisGFP1, a variant of IrisFP (24,34,35). After co-transfection with desmin expression constructs under the same constitutive promoter, filament formation was ana- lyzed in living cells by dual color PALM.…”

Genetic evidence for a microtubule-capture mechanism during polar growth of Aspergillus nidulans

“…S4A, Movie 4). In addition to eGFP fusions, we generated a fusion with photoconvertible mEosFPthermo (Wiedenmann et al, 2004(Wiedenmann et al, , 2011, which allows for analysis using super-resolution microscopy, such as photoactivated localization microscopy (PALM; for a review of super-resolution microscopy, see Patterson et al, 2010). Super-resolution single-particle-tracking analysis of MigA-mEosFPthermo (MigA tagged at the C-terminus) clusters provided essentially background-free images and showed localization of single MigA clusters at growing and retracting MTs (Fig.…”