5H87: Crystal Structure Of Mrojoa Mutant - P63h - W143s

Red fluorescent proteins (RFPs) are used extensively in chemical biology research as fluorophores for live cell imaging, as partners in FRET pairs, and as signal transducers in biosensors. For all of these applications, brighter RFP variants are desired. Here, we used rational design to increase the quantum yield of monomeric RFPs in order to improve their brightness. We postulated that we could increase quantum yield by restricting the conformational degrees of freedom of the RFP chromophore. To test our hypothesis, we introduced aromatic residues above the chromophore of mRojoA, a dim RFP containing a pi-stacked Tyr residue directly beneath the chromophore, in order to reduce chromophore conformational flexibility via improved packing and steric complementarity. The best mutant identified displayed an absolute quantum yield increase of 0.07, representing an over 3-fold improvement relative to mRojoA. Remarkably, this variant was isolated following the screening of only 48 mutants, a library size that is several orders of magnitude smaller than those previously used to achieve equivalent gains in quantum yield in other RFPs. The crystal structure of the highest quantum yield mutant showed that the chromophore is sandwiched between two Tyr residues in a triple-decker motif of aromatic rings. Presence of this motif increases chromophore rigidity, as evidenced by the significantly reduced temperature factors compared to dim RFPs. Overall, the approach presented here paves the way for the rapid development of fluorescent proteins with higher quantum yield and overall brightness.
PDB ID: 5H87Download
MMDB ID: 136090
PDB Deposition Date: 2015/12/23
Updated in MMDB: 2016/03
Experimental Method:
x-ray diffraction
Resolution: 2.24  Å
Source Organism:
Similar Structures:
Biological Unit for 5H87: monomeric; determined by author and by software (PISA)
Molecular Components in 5H87
Label Count Molecule
Protein (1 molecule)
Mrojoa Fluorescent Protein
Molecule annotation
* Click molecule labels to explore molecular sequence information.

Citing MMDB