Computational modeling of a new fluorescent biosensor for caspase proteolytic activity improves dynamic range.
The class of fluorescence resonance energy transfer (FRET) protein biosensors that are useful for measuring protease activity is composed of a tandem fusion of yellow fluorescent protein (YFP), a cleavage recognition sequence, and cyan fluorescent protein (CFP). The dynamic range of these FRET-based protein biosensors is often weak, but applications such as high throughput drug screening require stronger dynamic ranges. Using the biosensor for the caspase-3 protease as an example, here we showed a computational approach to improve the FRET dynamic range based on the atomic structure of caspase-3 bound to its inhibitor. This result was verified from our experiments where the FRET dynamic range improved by at least 60% on average in both in vitro and in vivo contexts. In concept, the same strategy can be applied to improve dynamic range of other FRET-based protein biosensors for protease activity where there exist solved atomic structures for protein complexes.
Chiang
Jason Jui-Hsuan
JJ
Department of Electrical and Computer Engineering (ECE), Institute of Biomaterials and Biomedical Engineering (IBBME), University of Toronto, Toronto, ON M5S 3G4, Canada. jjason.chiang@utoronto.ca
Truong
Kevin
K
D053148:Caspase 3; D020169:Caspases / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D003198:Computer Simulation; D017076:Computer-Aided Design; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods; D008956:Models, Chemical; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D010447:Peptide Hydrolases; D053148:Caspase 3; D020169:Caspases