An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane

R. P. Baker, S. Urban

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Intramembrane proteases are an ancient and diverse group of multispanning membrane proteins that cleave transmembrane substrates inside the membrane to effect a wide range of biological processes. As proteases, a clear understanding of their function requires kinetic dissection of their catalytic mechanism, but this is difficult to achieve for membrane proteins. Kinetic measurements in detergent systems are complicated by micelle fusion/exchange, which introduces an additional kinetic step and imposes system-specific behaviors (e.g., cooperativity). Conversely, kinetic analysis in proteoliposomes is hindered by premature substrate cleavage during coreconstitution, and lack of methods to quantify proteolysis in membranes in real time. In this chapter, we describe a method for the real-time kinetic analysis of intramembrane proteolysis in model liposomes. Our assay is inducible, because the enzyme is held inactive by low pH during reconstitution, and fluorogenic, since fluorescence emission from the substrate is quenched near lipids but restored upon proteolytic release from the membrane. The precise measurement of initial reaction velocities continuously in real time facilitates accurate steady-state kinetic analysis of intramembrane proteolysis and its inhibition inside the membrane environment. Using real data we describe a step-by-step strategy to implement this assay for essentially any intramembrane protease.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages229-253
Number of pages25
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume584
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Alzheimer's disease
  • Cancer
  • Cell signaling
  • ER-associated degradation
  • Gamma-secretase
  • Malaria
  • Membrane protein
  • Parkinson's disease
  • Presenilin
  • Protease
  • Regulated intramembrane proteolysis
  • Site-2 protease

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Baker, R. P., & Urban, S. (2017). An Inducible Reconstitution System for the Real-Time Kinetic Analysis of Protease Activity and Inhibition Inside the Membrane. In Methods in Enzymology (pp. 229-253). (Methods in Enzymology; Vol. 584). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2016.10.025