Abstract
Biosensors based on Förster Resonance Energy Transfer (FRET) between fluorescent protein mutants have started to revolutionize physiology and biochemistry. However, many types of FRET biosensors show relatively small FRET changes, making measurements with these probes challenging when used under sub-optimal experimental conditions. Thus, a major effort in the field currently lies in designing new optimization strategies for these types of sensors. Here we describe procedures for optimizing FRET changes by large scale screening of mutant biosensor libraries in bacterial colonies. We describe optimization of biosensor expression, permeabilization of bacteria, software tools for analysis, and screening conditions. The procedures reported here may help in improving FRET changes in multiple suitable classes of biosensors.
Highlights
FRET (Förster Resonance Energy Transfer) between mutants of the Green Fluorescent Protein GFP and its variants has become a preferred mode to monitor protein interactions and conformational change in many different applications [1,2]
The goal was to select sensors with large FRET changes out of a library of diversified sensors that would encompass a broad range of FRET ratio values both under resting conditions and in ligand bound state
These sensor libraries were transformed into bacteria and the bacteria were plated onto LB agar plates
Summary
FRET (Förster Resonance Energy Transfer) between mutants of the Green Fluorescent Protein GFP and its variants has become a preferred mode to monitor protein interactions and conformational change in many different applications [1,2]. Changes in FRET within these sensors rely on alterations of the distance and the orientation of the donor and acceptor fluorescent protein to each other. Targeted protein engineering has been used to increase these FRET changes by altering linker sequences between fluorophores and ligand binding domains, mutating hinge residues within domains, or incorporating combinations of fluorescent proteins with various circular permutations, providing a range of angles and orientations between fluorophores to choose from. Optimizing such changes has been time consuming and labor intensive. Libraries consisted of combinations of circularly permutated donor and acceptor fluorophores [13,14,15,16,17] and/or choices of extended, flexible or rigid linkers [18,19,20,21], typically comprising not more than a few hundred mutant sensors
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