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

静电控制下膜界面的蛋白质折叠和错误折叠：结合振动斯塔克效应、表面增强和纳米红外光谱

• 批准号: 500707750
• 负责人: Dr. Jacek Artur Kozuch
• 金额: --
• 依托单位: Institut für Experimentalphysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/500707750?language=en
• 关键词: Protein; Folding; Misfolding; Membrane; Interfaces

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.

自从发现蛋白质和肽中的内在无序以来，很明显，结构以及结构的缺乏都可以有目的地在氨基酸序列中编码，并且需要进一步的结合伴侣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，并可导致蛋白质聚集，如在阿尔茨海默氏症或2型糖尿病中观察到的，这是痴呆症的主要原因。在这些蛋白质聚集疾病中，肽β-淀粉样蛋白（Aβ）或胰岛淀粉样蛋白多肽（IAPP）表现出在膜界面处向原纤维的错误折叠增强，导致功能障碍甚至细胞死亡。已经表明膜静电在肽向淀粉样蛋白形成的结构转变中起特定作用：取决于膜组成，特别是带负电荷的脂质的含量，肽经历广泛的多态行为，以适应各种α-螺旋和β-折叠构象。有趣的是，这些物质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，不仅如何脂质组成，而且如何跨膜静电势梯度可以引导这些多态性，疾病相关肽的折叠事件的轨迹。为了深入了解这个问题，我建议利用围绕先进的红外（IR）光谱方法的表面增强红外吸收（SEIRA）和纳米级红外光谱与系留双层脂质膜系统（tBLMs）和振动斯塔克效应（VSE）联络的实验策略。tBLM和SEIRA的组合将能够在不同组成的膜处和膜中原位监测Aβ和IAPP的折叠过程，并在跨膜电位的直接控制下。此外，利用分子生物学的工具，我们将把含有CN基团的非天然氨基酸引入Aβ和IAPP，这将使得能够使用VSE框架量化局部静电环境的变化。后者还将提供一种策略来鉴定是否形成了不同的多态性β-折叠淀粉样蛋白种类，否则使用IR光谱法可能难以辨别。最后，我们将扩展SEIRA-tBLM方法对纳米级红外光谱方法，利用原子力显微镜尖端作为天线，以检测低于衍射极限的非均质系统的红外光谱，即具有约100 nm的空间分辨率。30纳米。该项目的结果将有助于了解脂膜的特定静电条件如何引导多态性蛋白沿着其平坦的构象景观折叠或错误折叠的结构，在淀粉样蛋白疾病的背景下。