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

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.