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.
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
Jason Jui-Hsuan
JJ
Chiang
Kevin
K
Truong
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