An autoinhibited coiled-coil design strategy for split-protein protease sensors.
Proteases are widely studied as they are integral players in cell-cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000-fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus, these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.
Shekhawat
Sujan S
SS
Department of Chemistry & Biochemistry, University of Arizona, Tucson, Arizona 85721, USA.
Porter
Jason R
JR
Sriprasad
Akshay
A
Ghosh
Indraneel
I
D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D048888:Fireflies / Q000201:enzymology*; D049409:Luciferases, Firefly / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D011506:Proteins; D049409:Luciferases, Firefly; D001618:beta-Lactamases