Structural Insight into the Fluorescence Mechanism of GFP-like Proteins（RIKEN SPring-8 Center）Akihiro Kikuchi
Green fluorescent protein from Aequorea victoria (avGFP) is now a ubiquitous tool in thefields of molecular and cellular biology for the visualization of gene expression, protein
localization, and protein–protein interactions in living cells. That’s why the 2008 Nobel Prizefor Chemistry has been awarded to Profs Shimomura, Chalfie and Tsien for the discovery anddevelopment of the avGFP.

Jellyfish (Aequorea Victoria)

Overall structure of avGFP

Chromophore of avGFP

In recent years, interest has grown in development of avGFP variants that exhibit efficient
optical properties for more localized and precise studies of protein movement in vivo.
Successful examples include ‘Dronpa’ showing reversible photochromism and ‘Keima’showing a large Stokes’ shift. Accordingly, structural study is of particular importance for theunderstanding the basis of their optical properties.
So far, we have solved dozens of crystal structures of GFP-like proteins and achieved
house-made crystallization kits suitable for crystallization of newly cloned GFP-like proteins.
1) Finding of a novel three-ring chromophore
Monomeric Kusabira-Orange (mKO) emits orange light at apeak of 559 nm. We have solved its crystal structure at 1.65 Åresolution and found a novel three-ring chromophore. The
conjugated π-electron system of the three-ring chromophore wasmore extended than that of avGFP, but less extended than that
Chromophore of mKOof the Discosoma sp. red fluorescent protein (DsRed). The structural
finding is sufficient to account for the orange-emitting ability of mKO.
2) Retinal fine-tuning of emission color

Crystal structures of a cyan-emitting protein (KCy) and its variant have been
determined. They revealed that the chromophore (Ser62-OH) of KCy makes a
non-covalent hydrogen-bonding interaction with His38 residue, which seems to govern
chromophore polarization. On the based of the structures, we successfully constructed thevariants that display a small red-shift in emission spectra, compared to the wild-type.

Fine-tuning of peak maximum in fluorescence spectra
3) Structural flexibility

The structural basis for the photochromism in Dronpa was poorly understood because acrystal structure of the dark state has been elusive. Unclear electron density around thechromophore (3.0 Å resolution) of Dronpa in the dark state should indicate disorder,
namely, flexibility of the chromophore.

Electron density map of Dronpa of the bright state (left) and the dark state (right)
with superimposed atomic model of the chromophore and the surroundings
Therefore, we performed NMR analysis of Dronpa to find structural flexibility of theprotein in the dark state. In the study, we have demonstrated that the combination of
crystallography and NMR is a powerful approach for studying dynamics in proteins.
4) Factor for Stokes’ shift
We have crystallized and subsequently determined the crystal structure of Keima thatshows a large shift between the colors of light it absorbs and emits, i.e. large Stokes’ shift.
The possible mechanism for the large Stokes’ shift will be discussed.
Acknowledgment
The structural study of GFP-like proteins has been carried on in collaboration with Dr
Miyawaki’s group in RIKEN BSI.