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

Sujan S. Shekhawat, Jason R. Porter, Akshay Sriprasad, Indraneel Ghosh

Research output: Contribution to journalArticle

53 Citations (Scopus)

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 β-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.

Original languageEnglish (US)
Pages (from-to)15284-15290
Number of pages7
JournalJournal of the American Chemical Society
Volume131
Issue number42
DOIs
StatePublished - Oct 28 2009

Fingerprint

Peptide Hydrolases
Proteins
Sensors
Biosensing Techniques
Biosensors
Firefly Luciferases
Logic gates
Cell Cycle Checkpoints
Caspase 3
Cell death
Cells
Switches
Apoptosis
Geometry
Testing

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

An autoinhibited coiled-coil design strategy for split-protein protease sensors. / Shekhawat, Sujan S.; Porter, Jason R.; Sriprasad, Akshay; Ghosh, Indraneel.

In: Journal of the American Chemical Society, Vol. 131, No. 42, 28.10.2009, p. 15284-15290.

Research output: Contribution to journalArticle

Shekhawat, Sujan S. ; Porter, Jason R. ; Sriprasad, Akshay ; Ghosh, Indraneel. / An autoinhibited coiled-coil design strategy for split-protein protease sensors. In: Journal of the American Chemical Society. 2009 ; Vol. 131, No. 42. pp. 15284-15290.
@article{869d41ee757a420aa2e0a48fcd54da67,
title = "An autoinhibited coiled-coil design strategy for split-protein protease sensors",
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 β-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.",
author = "Shekhawat, {Sujan S.} and Porter, {Jason R.} and Akshay Sriprasad and Indraneel Ghosh",
year = "2009",
month = "10",
day = "28",
doi = "10.1021/ja9050857",
language = "English (US)",
volume = "131",
pages = "15284--15290",
journal = "Journal of the American Chemical Society",
issn = "0002-7863",
publisher = "American Chemical Society",
number = "42",

}

TY - JOUR

T1 - An autoinhibited coiled-coil design strategy for split-protein protease sensors

AU - Shekhawat, Sujan S.

AU - Porter, Jason R.

AU - Sriprasad, Akshay

AU - Ghosh, Indraneel

PY - 2009/10/28

Y1 - 2009/10/28

N2 - 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 β-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.

AB - 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 β-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.

UR - http://www.scopus.com/inward/record.url?scp=70350329253&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=70350329253&partnerID=8YFLogxK

U2 - 10.1021/ja9050857

DO - 10.1021/ja9050857

M3 - Article

C2 - 19803505

AN - SCOPUS:70350329253

VL - 131

SP - 15284

EP - 15290

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 42

ER -