Investigation of Molecular Dynamics of Substrate Transmembrane alpha-Helices by Solution and Solid-State NMR Spectroscopy

膜内蛋白水解是膜蛋白加工的重要机制，与疾病的发生有直接关系。这个过程是如何在亲脂环境中发生的，底物的序列或动态特征的哪些细节对蛋白质水解是必不可少的，这些问题目前还在争论中。在这个项目中，我们通过液体和固体核磁共振波谱研究了所选底物TM螺旋的结构和动力学要求，这是目前唯一的在原子细节上研究生物分子动力学的实验技术。我们将从两个模型TM螺旋开始：APP，一种研究得很好的γ分泌酶底物，和PINK1，菱形蛋白酶PARL的底物。在目标1中，我们将专注于TM结构域在溶液和膜模拟环境中的构象可塑性，特别关注与规范螺旋结构的偏差，并寻找与主构象以及相关动力学参数交换的低填充状态（隐藏状态）。我们将直接测量表征TM域的动态参数，如局部交换率和一系列膜模型中单个原子间向量的运动幅度。在目标2中，我们将研究两个TM结构域突变的影响。已知其中一些突变会损害蛋白质水解。我们将阐明它们如何改变TM螺旋的灵活性。在目标3中，我们将研究膜如何影响TM螺旋结构和动态特性。预期结果将与P7中进行的分子动力学模拟进行比较，并显示TM结构域动力学是否是蛋白酶识别和/或加工的相关因素。根据目标1-3的进展情况，将在P1/P2/P3中选定的新底物上应用方案和实验，以判断这些研究的总体有效性。

Intramembrane proteolysis is an important mechanism of membrane protein processing with immediate relevance for the development of diseases. How this process takes place in the lipophilic environment and which details of the sequence or dynamic features of the substrates are essential for proteolysis are questions that are currently under debate. In this project we investigate the structural and dynamical requirements of selected substrate TM helices by liquid and solid-state NMR spectroscopy, currently the only experimental techniques to study dynamics of biomolecules at atomic detail. We will begin with two model TM helices: APP, a well-studied gamma-secretase substrate, and PINK1, substrate of the rhomboid protease PARL. In Goal 1 we will concentrate on the conformational plasticity of the TM domains in solution and membrane-mimicking environments focusing in particular on deviations from the canonical helical structure and searching for lowly populated states (hidden states) in exchange with the main conformation as well as the related dynamical parameters. We will directly measure dynamical parameters characterizing the TM domains such as local exchange rates and motional amplitudes for individual interatomic vectors in a range of membrane models. In Goal 2 we will investigate the impact of mutations of the two TM domains. Some of these mutations are already known to impair proteolysis. We will clarify how they alter TM helix flexibility. In Goal 3 we will study how the membrane influences TM helix structural and dynamic properties. The expected results will be compared with molecular dynamics simulations conducted in P7 and show if the TM domain dynamics is a relevant factor for the protease recognition and/or processing. Depending on progress in goals 1-3, protocols and experiments will be applied on selected novel substrates identified in P1/P2/P3 to judge the general validity of these studies.