title: Activity Assays for Rhomboid Proteases.
abstract: Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KMand Vmaxto measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.
authors.affiliation: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
authors.forename: E
authors.initials: E
authors.lastname: Arutyunova
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: K
authors.initials: K
authors.lastname: Strisovsky
authors.affiliation: Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada. Electronic address: mlemieux@ualberta.ca.
authors.forename: M J
authors.initials: MJ
authors.lastname: Lemieux
mesh_terms: D020134:Catalytic Domain; D002462:Cell Membrane / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D059748:Proteolysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D010450:Endopeptidases
keywords: AarA; Artificial and natural substrates FRET; Catalytic efficiency; Catalytic turnover; GlpG enzymatic activity; In vivo and in vitro assays; Rhomboids; psTatA