Engineering fluorescent protein substrates for the AAA+ Lon protease.
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.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Matthew L
ML
Wohlever
Andrew R
AR
Nager
Tania A
TA
Baker
Robert T
RT
Sauer
D004926:Escherichia coli / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D007700:Kinetics; D049070:Protease La / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015202:Protein Engineering; D017510:Protein Folding; D059748:Proteolysis; D013379:Substrate Specificity
D049452:Green Fluorescent Proteins; D049070:Protease La