Substrate Transmembrane Helices: Conformational Flexibility and Recognition by an Enzyme

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A primary event in substrate selection is its recognition by the cognate intramembrane protease. We posit that recognition as well as subsequent cleavage of a substrate is crucially influenced by the conformational flexibility of its transmembrane helix, an aspect that is currently unexplored! In Goal 1, we will investigate transmembrane helix motions in a comparative study of substrates and non-substrates for different intramembrane proteases (gamma-secretase, rhomboid PINK1, signal peptide peptidases). To this end, we will apply techniques primarily involving mass spectrometry to study local unfolding around helical cleavage regions as well as global conformational changes of substrate transmembrane helices. Biologically relevant patterns of helix flexibility will be uncovered by comparing the impacts of point mutations on flexibility and on substrate binding and cleavage. Apart from these applied aspects, a systematic comparison of primary structure and transmembrane helix dynamics will yield important new insights into the way both are connected. In Goal 2, we will develop FRET-based and biochemical assays to study the interaction of the C99 TM domain and the substrate-binding presenilin TM domain in order to identify substrate amino acids critical for binding. Such assays will also enable us to investigate the potential role of the C99 TM domain dimer in substrate/enzyme interaction. Another important question will adress the regulation of substrate/enzyme interaction by lipids by connecting the interaction in different lipid environments to FRET-based analysis of substrate/lipid interaction to be studied in parallel.