Photoconvertible fluorescent proteins (pcFPs) constitute a large group of fluorescent proteins related to green fluorescent protein (GFP) that, when exposed to blue light, bear the capability of irreversibly switching their emission color from green to red. Not surprisingly, this fascinating class of FPs has found numerous applications, in particular for the visualization of biological processes. A detailed understanding of the photoconversion mechanism appears indispensable in the design of improved variants for applications such as super-resolution imaging. In this article, recent work is reviewed that involves using pcFPs as a model system for studying protein dynamics. Evidence has been provided that the evolution of pcFPs from a green ancestor involved the natural selection for altered dynamical features of the beta-barrel fold. It appears that photoconversion may be the outcome of a long-range positional shift of a fold-anchoring region. A relatively stiff, rigid element appears to have migrated away from the chromophore-bearing section to the opposite edge of the barrel, thereby endowing pcFPs with increased active site flexibility while keeping the fold intact. In this way, the stage was set for the coupling of light absorption with subsequent chemical transformations. The emerging mechanistic model suggests that highly specific dynamic motions are linked to key chemical steps, preparing the system for a concerted deprotonation and β-elimination reaction that enlarges the chromophore’s π-conjugation to generate red color.

Rubisco enzymes play central roles in carbon fixation, with potential importance in biotechnology, but have eluded a full description of their multistep assembly and function. A new article describes the fascinating discovery that some archaeal Rubiscos contain a built-in assembly domain inserted into an otherwise canonical Rubisco fold, providing a tremendous expansion of our understanding of the diversity of naturally occurring Rubiscos.

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.