Single molecule IR nanoscopy on solid-supported membrane proteins

这一系统项目的目标是对单个膜蛋白的分子结构和动力学进行纳米研究。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)相结合，为单分子中红外吸收光谱提供一个很好的平台。微结构的金表面，具有很强的红外吸收增强作用，将作为完整的膜蛋白的固体支撑。由于传统的红外显微光谱学缺乏横向分辨率和灵敏度，读数将进一步增强，并通过金属化原子力显微镜(AFM)尖端的横向分辨率来实现。该方法学应适用于膜生物学中两个不同的相关挑战。在进行单分子红外光谱的尝试中，我们的目标是记录酰胺I范围内时间分辨的中红外吸收变化，以检索表面拴系膜蛋白的结构信息。我们的中红外sSNOM将扩展到水环境中。将进行拉力实验，以量化单个膜蛋白展开事件发生所需的力，同时记录瞬时的中IR吸收变化，以产生展开过程的补充结构信息。对微生物视紫红质的时间分辨研究将导致单个蛋白质在功能上的结构变化。这些单分子实验将得到对固体支撑膜和自组装有机聚合物表面的纳米FTIR(纳米傅立叶变换红外)光谱研究的补充。利用飞秒中红外激光系统作为宽带红外源，记录FTIR近场光谱，同时AFM针尖扫描表面，以横向分辨率&lt；30 nm产生膜的化学信息。光谱记录应扩展到对嵌入在浸泡在水环境中的固体支撑生物膜中的视紫红质的光诱导差异纳米光谱。通过这些手段，将建立一种化学显微镜来在纳米尺度上确定生物膜的功能和横向异质性。此外，可以在单振动水平上追踪单个蛋白质的(去)折叠途径。

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.