Development and Application of Proton-Detected Solid-State NMR Spectroscopy for Elucidation of Membrane Protein Function

固体核磁共振在固体蛋白质，尤其是淀粉样蛋白和膜蛋白的结构和动力学表征方面具有越来越重要的意义。传统的方法完全依赖于非质子共振。最近，作为一种重要的补充，质子探测本身具有比13C高8倍的灵敏度，引起了人们的极大兴趣。固态核磁共振中1H化学位移的可用性归功于最近技术和方法的发展，我的贡献在其中发挥了主要作用。我们已经将质子用于各种现在流行的技术目的。然而，尽管质子/氢在所有细胞过程中都发挥着无处不在的作用，但在探索蛋白质功能方面，几乎没有利用氢离子信息的可及性。对于膜蛋白来说尤其如此，膜蛋白是固态核磁共振主要目标中的一类重要蛋白质。在以某种方式依赖于蛋白质与质子或氢的相互作用的无数生物过程中，我们的重点是两类具有代表性的膜蛋白，电压传感器和整膜蛋白酶。电压传感器（VS）是不同离子通道和酶的元件，特别是在调节神经元膜电位方面具有广泛的意义。我们将对一个小的、有代表性的VS进行研究，其中的原子结构、电压传感和氢导电机制在很大程度上是难以捉摸的，包括有望代表导电或不导电状态的突变体，用核磁共振方法表征质子的行为。因此，我们将阐明质子电导的机理特征和电压传感中依赖电位的构象变化。整膜蛋白酶是通过调节膜内蛋白水解（RIP）参与多种信号通路的酶。这一共同机制在信号传导、转录调控（如脂质生物合成）和淀粉样前体蛋白（APP）等蛋白质的裂解中发挥着重要作用。聚焦于一个小的，具有代表性的蛋白酶，我们将评估一般意义的问题，如结构域迁移，底物进入，以及水通道进入活性位点，埋在膜的疏水空间。在这两种情况下，对原子细节机制的理解取决于溶剂与水暴露侧链的相互作用、质子化状态、轨迹和表征质子/水转移的物理化学性质的详细信息，以及脂质环境中的一般结构和动力学参数。凭借我在氢检测固态核磁共振和膜蛋白的开发和应用方面的长期专业知识，本案例研究将在其生物学背景下对质子进行表征，这将为深入了解基本膜蛋白功能铺平道路，并为未来的结构生物学提供方法学基础。

Solid-state NMR is of ever-growing significance for characterization of structure and dynamics in solid proteins, most eminently amyloids and membrane proteins. Traditional methods have purely relied on non-proton resonances. Recently, as a significant complement, proton detection with intrinsically eightfold higher sensitivity compared with 13C has gained enormous interest. The availability of 1H chemical shifts in solid-state NMR owes to recent technical and methodological developments, for which my contributions have played a major role.We have employed protons for various now popular technical purposes. However, despite the ubiquitous role of protons/hydrogens in all cellular processes, accessibility of 1H information has hardly been exploited with respect to exploration of protein functionality. This is particularly true for membrane proteins, an important class of proteins among the main targets of solid-state NMR. Out of the innumerable biological processes hinging on the interplay of proteins with protons or hydrogens in some way, our focus is on two representative classes of membrane proteins, voltage sensors and integral-membrane proteases.Voltage sensors (VS) are elements of different ion channels and enzymes of broad significance particularly for regulating neuronal membrane potentials. We will subject a small, representative VS, of which atomic structure, voltage sensing, and 1H-conducting mechanism are largely elusive, including mutants expected to represent conducting or non-conducting states, to NMR methods characterizing proton behavior. As such, we will elucidate mechanistic features of proton conductance and the potential-dependent conformational changes in voltage sensing.Integral-membrane proteases are enzymes involved in several signaling pathways through regulated intra-membrane proteolysis (RIP). This common mechanism plays a prominent role in signaling, transcriptional regulation, for example of lipid biosynthesis, and in cleavage of proteins like the Amyloid Precursor Protein (APP). Focusing on a small, representative protease, we will assess questions of general significance, like domain mobility, substrate entry, and water channeling into the active site, buried in the hydrophobic space of the membrane.In both cases, understanding of the mechanism with atomic detail hinges on detailed information about solvent interactions with the water-exposed side chains, protonation states, trajectories, and physicochemical properties characterizing proton/water transfers in addition to the general structural and dynamics parameters in a lipid environment.Relying on my long-standing expertise in development and application of 1H-detected solid-state NMR and membrane-proteins, this case study towards characterization of protons in their biological context will pave the way for an unprecedented level of insight into basic membrane protein functionality and provide methodological groundwork for future structural biology.