Single molecule IR nanoscopy on solid-supported membrane proteins

这是一个有条理的项目的目标，对单个膜蛋白的分子结构和动力学进行纳米级研究。我们将使用表面增强红外吸收光谱（SEIRAS）和散射型扫描近场光学显微镜（sSNOM）的组合，为单分子中红外吸收光谱提供非常适合的平台。微结构的金表面，定制表现出强烈的红外吸收增强，将作为一个坚实的支持，以整合膜蛋白。由于缺乏横向分辨率和灵敏度的常规红外显微光谱，读出将进一步增强和横向解决的金属化原子力显微镜（AFM）针尖的顶点。该方法应适用于膜生物学中两种不同的相关挑战。在尝试进行单分子红外光谱，我们的目标是记录时间分辨中红外吸收变化的酰胺I范围内检索的表面束缚膜蛋白的结构信息。我们的中红外sSNOM应扩展到水性环境。将进行牵拉实验，以量化发生单个膜蛋白解折叠事件所需的力，同时记录瞬时中红外吸收变化，以产生解折叠过程的互补结构信息。对微生物视紫红质的时间分辨研究将导致单个蛋白质的功能相关结构变化。这些单分子实验将通过对固体支撑膜和自组装有机聚合物表面的nanoFTIR（纳米傅里叶变换红外）光谱研究来补充。飞秒中红外激光系统将被用作宽带红外光源，以记录FTIR近场光谱，同时AFM针尖扫描表面，以产生横向分辨率< 30 nm的膜的化学信息。光谱记录应扩展到光诱导差异纳米光谱的视紫红质嵌入在固体支撑的生物膜浸泡在水环境中。通过这些手段，将建立一个化学显微镜，以确定纳米尺度上的生物膜的功能和横向异质性。此外，单个蛋白质的（解）折叠途径可以在单振动水平上追踪。

It is the goal of this methodical project to perform nanoscopic studies on the molecular structure and dynamics of individual membrane proteins. We will use a combination of surface-enhanced infrared absorption spectroscopy (SEIRAS) and scattering-type scanning near-field optical microscopy (sSNOM), providing a well-suited platform for single molecule mid-IR absorption spectroscopy. Micro-structured gold surfaces, tailored to exhibit strong IR absorption enhancement, will serve as a solid support to integral membrane proteins. Due to the lack of lateral resolution and sensitivity of conventional IR microspectroscopy, the readout will be further enhanced and laterally resolved by the apex of a metalized atomic force microscope (AFM) tip. The methodology shall be applied to two different pertinent challenges in membrane biology. In an attempt to perform single molecule IR spectroscopy, we aim at recording time-resolved mid-IR absorption changes in the amide I range to retrieve structural information of surface-tethered membrane proteins. Our mid-IR sSNOM shall be extended to aqueous environments. Pulling experiments will be conducted to quantify the force required for an individual membrane protein unfolding event to occur while simultaneously recording transient mid-IR absorption changes to yield complementary structural information of the unfolding process. Time-resolved studies on microbial rhodopsins will result in functionally relevant structural changes of individual proteins. These single molecule experiments will be complemented by nanoFTIR (nano Fourier-transform infrared) spectroscopic studies on solid-supported membranes and self-assembled organic polymer surfaces. A fs mid-IR laser system will be utilized as a broadband IR source to record FTIR near-field spectra while the AFM tip is scanning the surface to yield chemical information of membranes at a lateral resolution < 30 nm. Spectral recordings shall be extended to light-induced difference nanospectroscopy on rhodopsins embedded in solid-supported biomembranes immersed in aqueous environments. By these means, a chemical microscope will be established to determine functionality and lateral heterogeneity in biomembranes on the nm scale. Moreover, the (un-)folding pathway of individual proteins can be traced on the level of single vibrations.