An autoinhibited coiled-coil design strategy for split-protein protease sensors

J Am Chem Soc. 2009 Oct 28;131(42):15284-90. doi: 10.1021/ja9050857.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biosensing Techniques / methods*
  • Fireflies / enzymology*
  • Luciferases, Firefly / analysis*
  • Luciferases, Firefly / chemistry
  • Luciferases, Firefly / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteins / chemistry*
  • Proteins / metabolism
  • Substrate Specificity
  • beta-Lactamases / analysis*
  • beta-Lactamases / chemistry
  • beta-Lactamases / metabolism

Substances

  • Proteins
  • Luciferases, Firefly
  • beta-Lactamases