Abstract
While several intramembrane cleaving proteases (iCliPs), enzymes that carry out proteolysis reactions within the membrane interior, have had their structures solved, basic biochemical questions such as what determines a substrate and how cleavage site is dictated remain. However, our inability to monitor enzyme-substrate interactions in a lipid environment is the dominant factor limiting our understanding of the central questions associated with these enzymes. Specifically, while there are many excellent techniques for the characterization of membrane proteins, many of them are only amenable to steady state measurements, require removal from the membrane, or are too low resolution to offer detailed information about changes in structure and environment. Here we offer a new method by which to observe structural fluctuations of the enzyme and the substrate during cleavage reactions within a membrane environment: isotope-assisted vibrational interrogation of bilayer-embedded systems (iVIBE). Deep UV resonance Raman (DUVRR) spectroscopy has previously been used to observe structural and environmental changes in both soluble and membrane proteins. Now, using an isotopically labeled protease and an unlabeled substrate we can resolve the spectral responses from each protein, allowing us to observe the cleavage reaction over time and determine the binding site, or structural fate of the substrate.
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