Substrate Transmembrane Helices: Conformational Flexibility and Recognition by an Enzyme

底物选择的一个主要事件是它被同源的膜内蛋白酶识别。我们假设底物的识别以及随后的切割受到其跨膜螺旋构象灵活性的关键影响，这一方面目前还没有被探索！在目标1中，我们将通过对不同膜内蛋白酶(伽马分泌酶、菱形PINK1、信号肽酶)的底物和非底物的比较研究来研究跨膜螺旋运动。为此，我们将应用主要涉及质谱学的技术来研究螺旋裂解区域周围的局部展开以及底物跨膜螺旋的整体构象变化。通过比较点突变对柔韧性以及底物结合和切割的影响，将揭示螺旋柔韧性的生物学相关模式。除了这些应用方面，对一级结构和跨膜螺旋动力学的系统比较将对两者的连接方式产生重要的新见解。在目标2中，我们将发展基于FRET的分析和生化分析来研究C99 TM结构域和底物结合的Presenlin TM结构域的相互作用，以确定对结合至关重要的底物氨基酸。这样的检测还将使我们能够研究C99TM结构域二聚体在底物/酶相互作用中的潜在作用。另一个重要的问题是通过将不同脂质环境中的底物/酶相互作用与基于FRET的底物/脂类相互作用的分析联系起来进行平行研究，从而涉及到脂类对底物/酶相互作用的调节。

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.