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Engineering fluorescent protein substrates for the AAA+ Lon protease.

Matthew L Wohlever1, Andrew R Nager, Tania A Baker

  • 1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Protein Engineering, Design & Selection : PEDS
|January 30, 2013
PubMed
Summary

Escherichia coli Lon protease degrades proteins using specific tags. Researchers developed circularly permuted GFP variants that Lon efficiently degrades, enabling new high-throughput assays for Lon activity.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteolysis

Background:

  • AAA+ proteases, like E. coli Lon, degrade proteins by recognizing peptide degrons.
  • Standard green fluorescent protein (GFP) variants are poorly degraded by Lon, limiting assay development.

Purpose of the Study:

  • To engineer novel substrates for efficient degradation by E. coli Lon.
  • To develop robust in vitro and in vivo assays for Lon protease activity.

Main Methods:

  • Construction and testing of circularly permuted GFP variants with N-terminal β20 or C-terminal sul20 degrons.
  • Monitoring substrate unfolding and degradation via loss of fluorescence.
  • Assessing Lon degradation kinetics in vitro and proteolysis in vivo.

Main Results:

  • Circularly permuted GFP variants with appended degrons are robustly unfolded and degraded by Lon.
  • Non-permuted GFP-sul20 shows very slow degradation due to inefficient degron extraction.
  • The engineered substrates facilitate high-throughput kinetic and in vivo assays.

Conclusions:

  • Circularly permuted GFP substrates overcome Lon's degradation limitations.
  • These novel substrates enable convenient and efficient monitoring of Lon protease activity.