Single molecule IR nanoscopy on solid-supported membrane proteins

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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.