Brighter Red Fluorescent Proteins by Rational Design of Triple-Decker Motif

Area of Science:

• Biochemistry
• Chemical Biology
• Structural Biology

Background:

• Red fluorescent proteins (RFPs) are crucial tools in chemical biology for live cell imaging, FRET pairs, and biosensors.
• Brighter RFP variants are highly sought after to improve the sensitivity and capabilities of these applications.

Purpose of the Study:

• To rationally design and engineer brighter monomeric red fluorescent proteins (mRFPs) with increased quantum yield.
• To investigate the structural basis for enhanced quantum yield through chromophore restriction.

Main Methods:

• Rational design approach by introducing aromatic residues to restrict chromophore conformational freedom.
• Mutagenesis of the mRojoA RFP and screening of a small library of variants.
• X-ray crystallography to determine the structure of high quantum yield mutants.

Main Results:

• Identified a novel mRFP variant with an absolute quantum yield increase of 0.07, over 3-fold improvement compared to mRojoA.
• Achieved significant quantum yield gains with a library size orders of magnitude smaller than previously reported.
• Crystal structure revealed a triple-decker aromatic motif sandwiching the chromophore, increasing its rigidity.

Conclusions:

• Restricting chromophore conformational flexibility via rational design is an effective strategy for rapidly developing brighter RFPs.
• The identified triple-decker motif enhances chromophore rigidity and quantum yield.
• This approach accelerates the development of fluorescent proteins with superior brightness for biological research.