Protein Folding and Misfolding at Membrane Interfaces under Electrostatic Control: Combining Vibrational Stark Effect, Surface-Enhanced, and Nano-Infrared Spectroscopy

自从蛋白质和多肽的内在无序被发现以来，很明显，结构和结构的缺失都可以在氨基酸序列中有目的地编码，并且需要更多的结合伙伴来诱导无序到有序的转变。然而，这具有严重错误折叠的高风险，并可能导致蛋白质聚集，正如在阿尔茨海默氏症或2型糖尿病中观察到的那样，这是痴呆症的主要原因。在这些蛋白质聚集性疾病中，β-淀粉样多肽(A-β)或胰岛淀粉样多肽(IAPP)显示出在膜界面向纤维错误折叠的增强，导致功能障碍甚至细胞死亡。膜静电学被认为在多肽向淀粉样蛋白形成的结构转变中起着特殊的作用：取决于膜的组成，特别是负电荷脂类的含量，多肽经历了广泛的多态行为，适应各种α-螺旋和β-折叠构象。有趣的是，这些物种中的一些在早期形成跨膜α-螺旋通道结构，并干扰膜的静电特性。这就提出了一个问题，不仅是脂质成分如何，而且跨膜静电势梯度如何引导这些多态的、与疾病相关的多肽的折叠事件的轨迹。为了深入了解这个问题，我建议利用一种以先进的红外光谱方法为中心的实验策略，即表面增强红外吸收(SEIRA)和纳米级红外光谱，并与栓系双层类脂膜系统(TBLMS)和振动斯塔克效应(VSE)相结合。TBLMS和SEIRA的结合将能够在跨膜电位的直接控制下，在不同组成的膜上和在膜中原位监测Aβ和IAPP的折叠过程。此外，利用分子生物学的工具，我们将把含CN基团的非天然氨基酸引入到Aβ和IAPP中，这将使我们能够利用VSE的框架来量化局部静电环境的变化。后者还将提供一种策略来识别是否形成了不同的多态β-Sheet淀粉样蛋白物种，否则使用红外光谱很难区分这些物种。最后，我们将把Seira-tBLM方法扩展到纳米尺度的红外光谱方法，这种方法利用原子力显微镜尖端作为天线来探测低于衍射限的非均相体系的红外光谱，即空间分辨率约为30 nm。该项目的结果将有助于理解在淀粉样蛋白疾病的背景下，脂膜上的特定静电条件如何引导多态蛋白质沿着其平坦的构象景观朝向折叠或错误折叠的结构。

Ever since the discovery of the intrinsic disorder in proteins and peptides, it became clear that both structure as well as the lack of structure can be purposefully encoded in the amino acid sequence, and further binding partners are required to induce disorder-to-order transitions. However, this bears a high risk of severe misfolding and can lead to protein aggregates as observed in Alzheimer’s or in Type 2 Diabetes, which are main causes of dementia. In these protein aggregation diseases, the peptides β-amyloid (Aβ) or islet amyloid polypeptide (IAPP) demonstrate enhanced misfolding towards fibrils at membrane interfaces, leading to dysfunction or even cell death. Membrane electrostatics have been suggested to play a particular role in the structural transitions of the peptides towards amyloid formation: depending on the membrane composition, in particular the content of negatively charged lipids, the peptides undergo an extensive polymorphic behavior adapting various α-helical and β-sheet conformations. Interestingly, some of these species form transmembrane α-helical channel structures early on and interfere with the electrostatic properties of the membrane. This raises the question of not only how lipid composition but also how the transmembrane electrostatic potential gradient can guide the trajectory of folding events of these polymorphic, disease-relevant peptides. To provide insight into this question, I propose to utilize an experimental strategy centered around the advanced infrared (IR) spectroscopic methods of surface-enhanced IR absorption (SEIRA) and nanoscale IR spectroscopy in liaison with tethered bilayer lipid membrane systems (tBLMs) and the vibrational Stark effect (VSE). The combination of tBLMs and SEIRA will enable to monitor the folding processes of Aβ and IAPP in-situ at and in membranes of various compositions and under the direct control of the transmembrane potential. Furthermore, using tools from molecular biology, we will introduce CN group-containing non-natural amino acids into Aβ and IAPP, which will enable to quantify changes in local electrostatic environments using the framework of the VSE. The latter will also provide a strategy to identify if different polymorphic β-sheet amyloid species have formed, which otherwise can be difficult to discern using IR spectroscopy. Lastly, we will extend the SEIRA-tBLM approach towards nanoscale IR spectroscopic methods, which utilize atomic force microscopy tips as antennas to detect IR spectra of heterogeneous systems below the diffraction limit, that is with a spatial resolution of ca. 30 nm. The results of this project will contribute to the understanding of how specific electrostatic conditions at lipid membranes can steer polymorphic proteins along their flat conformational landscape towards folded or misfolded structures in the context of amyloid diseases.