Modulation of Fluorescent Protein Chromophore to Detect Protein Aggregation

Abstract

While green fluorescent protein (GFP) has countless applications in biology, the utilization of its chromophore outside the protein is rarely reported for other purposes, possibly due to its extremely low fluorescence. Herein, we structurally modulated FP chromophore to selectively report on the misfolding and aggregation of a protein-of-interest (POI) in both purified in vitro systems and live cells. These fluorophores remain non-fluorescent after conjugating to a folded POI, and emit fluorescence quantitatively in the misfolded and aggregated POI. The excitation and emission wavelengths are well aligned with common laser sources and filters for microscopic detections. We demonstrated their capability of detecting the aggregation of purified proteins in in vitro assays and Halo-tagged pathogenic proteins in mammalian cell lines. Our work not only provides the first fluorogenic tool to detect protein aggregation, but also opens a new avenue for fluorescent protein chromophore mimics. Support or Funding Information This work was supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface, Paul Berg Early Career Professorship, Lloyd and Dottie Huck Early Career Award. (a) Structural modulations of the chromophore in green fluorescent protein (HBI) to detect protein misfolding and aggregation. The fluorogenicity originates from restriction of non-radiative bond rotations in the non-polar and rigid micro-environment of misfolded and aggregated proteins. (b) Concept of using FP mimics to detect a protein-of-interest's misfolding and aggregation both in vitro and in vivo. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.