Some fluorescent proteins of the GFP (Green Fluorescent Protein) family, called "phototransformable fluorescent proteins", change their conformation when illuminated at specific wavelengths. They can then be switched "at will" between fluorescent and non-fluorescent forms. Phototransformable fluorescent proteins have been developed for a variety of applications, but especially as markers for advanced fluorescence microscopy, including super-resolution microscopy. They are used to visualize at high resolution and/or to follow in real time the molecular activity in the different cellular compartments. The fluorescent properties of these markers can be modified by mutagenesis of key residues located near the chromophore, the fluorescent entity at the heart of the protein. Current research in the field aims at developing improved fluorescent proteins that are less sensitive to the physico-chemical conditions that these proteins face when expressed.

The rational design of improved labels requires a good understanding of how these fluorescent proteins function at the atomic scale. Until now, X-ray crystallography has been the main technique used to understand how the chemical environment of the chromophore affects the photophysics of fluorescent proteins. However, crystallographic structures lack crucial information on the conformational dynamics, protonation states and hydrogen bonds of the chromophore and nearby amino acids, as well as their dependence on physicochemical conditions (pH, temperature, …).
In a recent study, IRIG researchers demonstrate that solution NMR spectroscopy, coupled with an in situ illumination device, allows access to this missing information, thus improving the mechanistic understanding of the functioning of these fluorescent markers. By studying rsFolder, a reversibly switchable green fluorescent protein they designed a few years ago, the researchers identified "the NMR signature" of the four distinct configurations of its p-HBI chromophore, corresponding to cis and trans isomers, each of which is either protonated or deprotonated at the benzylidene ring of the chromophore. The relative populations and interconversion kinetics of these four chromophore states are dependent on the pH of the sample and the composition of the buffer. These modify in a complex way the strength of the hydrogen bonds that contribute to stabilize the chromophore in the protein scaffold. The researchers also demonstrate the important role that histidine at position 149 of the protein plays in stabilizing the chromophore in its trans configuration at certain pH values.

These results can be exploited to design and test new phototransformable variants that are more robust to changes in the local cellular environment. This work also introduces NMR spectroscopy to the field of fluorescent protein research as a new tool to probe the populations and dynamics of different chromophore conformations under a variety of physiologically relevant conditions.



The prefixes cis and trans are used to specify the geometrical configuration of a molecule by specifying whether, with respect to the main chain of the described molecule, the main functional groups are located on the same side (cis) or on opposite sides (trans).

A large number of reversibly switchable fluorescent proteins have been designed from hydrozoan and anthozoan fluorescent protein sequences. All of these proteins share an 11-stranded β-barrel fold containing an endogenous 4-(p-hydroxybenzylidene)-5-imidazolinone (p-HBI) chromophore.