Far-red acclimating cyanobacterium as versatile source for bright fluorescent biomarkers

Area of Science:

• Biochemistry and Molecular Biology
• Biotechnology and Bioimaging

Background:

• Far-red and near-infrared fluorescent proteins are crucial for deep-tissue imaging but challenging to engineer.
• Cyanobacteria-derived biliproteins offer a promising alternative for novel chromophore development.

Purpose of the Study:

• To engineer novel bright far-red emitting chromoproteins from a thermophilic cyanobacterium's biliprotein (ApcF2).
• To develop a versatile toolbox of fluorescent labeling agents for various bioimaging applications.

Main Methods:

• Structure-guided, site-directed random and specific mutagenesis of ApcF2.
• Screening for bright far-red fluorescence.
• Characterization of engineered chromoproteins (BDFPs) in vitro and in vivo (mammalian cells, C. elegans).

Main Results:

• Generation of novel chromoproteins (BDFPs) that auto-catalytically bind biliverdin.
• BDFPs exhibit brightness, photostability, and fluorescence in mammalian cells and C. elegans.
• BDFPs tolerate harsh conditions (high temperature, low pH, chemical denaturation) and form useful tandem systems.

Conclusions:

• BDFPs represent a novel class of small, versatile, and robust fluorescent labeling tools.
• Engineered BDFPs offer significant advantages over existing biliproteins for diverse bioimaging techniques.
• These chromoproteins are suitable for advanced applications including FRET and super-resolution microscopy.