ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 363 (2007) 83–90 www.elsevier.com/locate/yabio 0003-2697/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.12.018 A Xuorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-speciWc small ubiquitin-like modiWer processing Sarah F. Martin a,1 , Neil Hattersley b,2 , Ifor D.W. Samuel a , Ronald T. Hay b , Michael H. Tatham b,¤,3 a Biophotonics Collaboration, School of Physics and Astronomy, University of St. Andrews, North Haugh, St. Andrews KY16 9SS, Scotland, UK b Centre for Interdisciplinary Research, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK Received 28 September 2006 Available online 4 January 2007 Abstract Dynamic modiWcation of proteins with the small ubiquitin-like modiWer (SUMO) aVects the stability, cellular localization, enzymatic activity, and molecular interactions of a wide spectrum of protein targets. We have developed an in vitro Xuorescence-resonance-energy- transfer-based assay that uses bacterially expressed substrates for the rapid and quantitative analysis of SUMO paralog-speciWc C-termi- nal hydrolase activity. This assay has applications in SUMO protease characterization, enzyme kinetic analysis, determination of SUMO protease activity in eukaryotic cell extracts, and high-throughput inhibitor screening. In addition, while demonstrating such uses, we show that the SUMO-1 processing activity in crude HeLa cell extracts is far greater than that of SUMO-2, implying that diVerential maturation rates of SUMO paralogs in vivo may be functionally signiWcant. The high degree of structural conservation across the ubiquitin-like pro- tein superfamily suggests that the general principle of this assay should be applicable to other post-translational protein modiWcation sys- tems.  2006 Elsevier Inc. All rights reserved. Keywords: SUMO; Ubiquitin-like modiWer; Sentrin Protease; FRET; Paralog speciWcity; C-terminal hydrolase Fluorescence resonance energy transfer (FRET) 4 is used in a multitude of biological applications including live cell imaging, analysis of protein–protein interactions, and pro- tein structure determination (see [1–3] for reviews). We have developed a FRET-based assay for the analysis of the cleavage of the peptide bond between the ubiquitin-like protein modiWers (ULPs) and their inhibitory peptides. We demonstrate this using two ULPs (SUMO-1 and SUMO-2) and show that SUMO processing activity can be accurately and reproducibly measured in both crude and puriWed sam- ples and that the assay can be applied to enzyme kinetic analysis, characterization of eukaryotic cell lysates, and high-throughput inhibitor screens. The posttranslational modiWcation of proteins with ubiquitin or molecules with ubiquitin-like sequences and structures has important regulatory consequences (see [4–6] for reviews). Higher eukaryotes are thought to express about 12 ubiquitin-like modiWers that are covalently attached to the -amino group of speciWc lysine residues on * Corresponding author. Fax: +44 1382 386302. E-mail address: M.Tatham@dundee.ac.uk (M.H. Tatham). 1 EPSRC Biophotonics platform funded. 2 BBSRC funded. 3 AICR funded. 4 Abbreviations used: ULP, ubiquitin-like protein modiWer, FRET, Xuo- rescence resonance energy transfer, SUMO, small ubiquitin-like modiWer; SUMO-1-FL, SUMO-1 full length (i.e., residues 1–101), SUMO-2-FL, SUMO-2 full length (i.e., residues 1–103), GFP, green Xuorescent protein; YFP, yellow Xuorescent protein, ECFP, enhanced cyan-Xuorescent pro- tein, SDS, sodium dodecyl sulfate, SENP, sentrin-speciWc protease; AMC, 7-amido-4-methylcoumarin, TEV, tobacco etch virus.