Time-resolved quantum cascade laser (QCL) IR spectroscopy to unravel membrane dynamics during protein activity

• Applicant: Professorin Dr. Karin Hauser
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 564132680

Time-resolved IR spectroscopy is a powerful label-free method to study biomolecular dynamics and interaction. By the introduction of quantum cascade lasers (QCL), an IR source was provided that is superior in spectral brilliance compared to FTIR spectroscopy yielding high sensitivity and data quality to analyze small spectral changes during the activity of membrane proteins. In this project, we will use the model membrane protein bacteriorhodopsin (BR), a light-driven proton pump whose photoreaction is IR-spectroscopically well characterized, in combination with photo-switchable artificial membranes to extend our knowledge on membrane dynamics, membrane function and site-specific membrane-protein interactions. In our previous step-scan FTIR studies, we could demonstrate that the environmental lipid membrane affects BR activity, i.e. changes in the proton translocation dynamics as well as the conformational dynamics of the protein. However, direct probing of the lipid vibrational modes and quantitative analyses were not feasible with FTIR as this method reaches its limits. Only the application of our home-built QCL-spectrometer enabled us to quantitatively analyze and correlate the lipid dynamics to the photoreaction of BR. As major focus of the project, we will explore the use of photo-switchable membranes to induce membrane dynamics in a controlled manner, identify specific lipid-protein interactions and monitor the impact on protein activity. Therefore, different membrane mimetics (vesicles and nanodiscs) will be characterized upon cw and pulsed photo-switching by time-resolved QCL-spectroscopy. Vesicles are commonly used membrane mimetics whereas nanodiscs provide insights into membrane lateral pressure. Various lipid modes will be evaluated as local reporters to probe different membrane regions. Reconstitution of BR into the photo-switchable membranes will allow to analyze quantitatively to what extent the induced membrane dynamics affect the proton transfer and conformation of BR. A main goal is to characterize membrane states during protein activity. We will gain a deeper understanding of the role of membrane dynamics for the function of membrane proteins in general. In long-term perspective, the knowledge from the model systems will be transferred to other membrane proteins and processes as e.g. protein insertion into membranes. The sensitivity of time-resolved QCL-spectroscopy will reveal the complex protein-membrane interplay with high molecular resolution.

• DFG Programme: Research Grants