Biochemical Characterization of Function and Structure of RseP, an Escherichia coli S2P Protease

• Published in: Methods in enzymology Vol. 584; p. 1
• Main Authors: Hizukuri, Y, Akiyama, K, Akiyama, Y
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: Endopeptidases - chemistry; Endopeptidases - metabolism; Escherichia coli - chemistry; Escherichia coli - enzymology; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Molecular Biology - methods; Signal Transduction; Structure-Activity Relationship; Substrate Specificity; Transcription Factors - chemistry; Thiol modification; In vivo cross-linking; Protein purification; Escherichia coli; RIP; Bacteria; Membrane; I-CLiP
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2016.09.044

Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σ protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.