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- Title
Engineering fluorescent protein substrates for the AAA+ Lon protease.
- Authors
Wohlever, Matthew L.; Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.
- Abstract
AAA+ proteases, such as Escherichia coli Lon, recognize protein substrates by binding to specific peptide degrons and then unfold and translocate the protein into an internal degradation chamber for proteolysis. For some AAA+ proteases, attaching specific degrons to the N- or C-terminus of green fluorescent protein (GFP) generates useful substrates, whose unfolding and degradation can be monitored by loss of fluorescence, but Lon fails to degrade appropriately tagged GFP variants at a significant rate. Here, we demonstrate that Lon catalyzes robust unfolding and degradation of circularly permuted variants of GFP with a β20 degron appended to the N terminus or a sul20 degron appended to the C terminus. Lon degradation of non-permuted GFP-sul20 is very slow, in part because the enzyme cannot efficiently extract the degron-proximal C-terminal β-strand to initiate denaturation. The circularly permuted GFP substrates described here allow convenient high-throughput assays of the kinetics of Lon degradation in vitro and also permit assays of Lon proteolysis in vivo.
- Subjects
PROTEOLYTIC enzymes; ESCHERICHIA coli; PROTEIN binding; DENATURATION of proteins; PEPTIDES; GREEN fluorescent protein
- Publication
PEDS: Protein Engineering, Design & Selection, 2013, Vol 26, Issue 4, p299
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzs105