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

固体核磁共振在描述固体蛋白质，尤其是淀粉样蛋白和膜蛋白的结构和动力学方面具有越来越重要的意义。传统的方法纯粹依赖于非质子共振。最近，作为一个重要的补充，质子探测以其固有的比13C高8倍的灵敏度获得了巨大的兴趣。固体核磁共振中1H化学位移的出现要归功于最近的技术和方法的发展，我的贡献对此起到了重要作用。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的作用无处不在，但在探索蛋白质功能方面，1H信息的可获得性几乎没有被利用。对于膜蛋白来说尤其如此，膜蛋白是固体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。电压传感器是不同离子通道的元件和具有广泛意义的酶，特别是在调节神经元膜电位方面。我们将对一个小的、有代表性的VS，其中的原子结构、电压传感和1H导电机制很难捉摸，包括预计代表导电或非导电状态的突变体，用核磁共振方法来表征质子的行为。因此，我们将阐明质子电导的机制特征和电压感知中依赖于电位的构象变化。完整膜蛋白水解酶是通过调节膜内蛋白分解(RIP)参与几个信号通路的酶。这种共同的机制在信号传递、转录调节等方面发挥着重要作用，例如脂质生物合成，以及对淀粉样前体蛋白(APP)等蛋白质的切割。聚焦于一个小的、有代表性的蛋白酶，我们将评估具有普遍意义的问题，如结构域迁移率、底物进入和水进入膜疏水空间中的活性部位。在这两种情况下，对原子细节机制的理解取决于关于溶剂与水暴露的侧链的相互作用、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，以及脂环境中的一般结构和动力学参数。依靠我在1H检测的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，这一针对质子生物学特征的案例研究将为深入了解基本的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.