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

固态核磁共振对于固体蛋白质，特别是淀粉样蛋白和膜蛋白的结构和动力学的表征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，作为一个重要的补充，质子检测与本质上八倍更高的灵敏度相比，13 C已经获得了巨大的兴趣。固体核磁共振氢化学位移的可用性应归功于最近的技术和方法的发展，我的贡献发挥了重要作用。我们使用质子为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的作用无处不在，但1H信息的可访问性几乎没有被用于探索蛋白质功能。这对于膜蛋白尤其如此，膜蛋白是固态NMR的主要目标中的一类重要蛋白质。在无数的生物过程中，蛋白质与质子或氢以某种方式相互作用，我们的重点是两类具有代表性的膜蛋白，电压传感器和整合膜蛋白酶。电压传感器（VS）是不同的离子通道和酶的元件，特别是调节神经元膜电位的广泛意义。我们将受到一个小的，有代表性的VS，其中原子结构，电压传感，和1H导电机制在很大程度上是难以捉摸的，包括突变体预计代表导电或非导电状态，核磁共振方法表征质子的行为。因此，我们将阐明质子电导和电位依赖的电压sensing.Integral-membrane蛋白酶的构象变化的机制功能，通过调节膜内蛋白水解（RIP）参与几个信号通路。这种常见机制在信号传导、转录调节（例如脂质生物合成）和蛋白质（如淀粉样前体蛋白（APP））的切割中起重要作用。以一个小的、有代表性的蛋白酶为重点，我们将评估具有普遍意义的问题，如结构域迁移、底物进入和水通道进入活性位点，埋在膜的疏水空间中。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、和物理化学性质表征质子/水转移除了一般的结构和动力学参数在脂质环境中.依靠我长期的专业知识，在开发和应用的1H-检测固态NMR和膜蛋白，本案例研究对质子在其生物学背景下的表征将铺平道路，为前所未有的水平的洞察基本膜蛋白质的功能，并提供方法基础，为未来的结构生物学。

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.