Ionic/non-ionic hybrid detergents for cryo-electron microscopy of membrane proteins

• Applicants: Dr. Tobias Raisch; Dr. Leonhard Hagen Urner
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Biological and Biomimetic Chemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 562810180

Membrane proteins are targets for 60% of current drugs and technologies to improve their structural investigation are urgently needed. State-of-the-art workflows for high-resolution structural investigations involve protein purification with detergents followed by the visualization of three-dimensional protein structures by cryo-electron microscopy (cryo-EM). Detergents that stabilize membrane proteins in solution often do not provide particle densities in vitrified ice that are sufficient for 3D-reconstruction of membrane protein complexes due to protein aggregation and unfolding at the water-air interface. To overcome this challenge, our proposal leverages a new detergent chemistry, i.e., ionic/non-ionic hybrid detergents, in combination with modern cryo-EM equipment and compelling membrane protein targets. Detergents with headgroups that contain solely ionic functional groups can modulate biomolecular interactions at water-air interfaces but denature membrane proteins already during purification. To uncouple desirable interfacial properties of detergents from protein denaturation, here we propose to fuse ionic and non-ionic functional groups to obtain hybrid detergents that stabilize membrane proteins in solution and reduce their interactions with water-air interfaces for improved particle densities in vitrified ice and 3D-reconstructions. To accomplish this aim, our project combines the expertise of Dr. Leonhard H. Urner (TU Dortmund University) in detergent science and studying interfacial properties of amphiphiles with Dr. Tobias Raisch’s (MPI Dortmund) expertise in structural biology and cryo-EM of compelling membrane protein targets. Our detergents will be synthesized and characterized in the Urner lab for their molecular and interfacial properties to enable the purification and cryo-EM analysis of intact membrane proteins in the Raisch lab. Only together, we will be able to evaluate our hypothesis that ionic/non-ionic hybrid detergents stabilize membrane proteins in solution and reduce their interactions with water-air interfaces for improved particle densities in vitrified ice. Following successful evaluation of our hypothesis, we will leverage our knowledge gain to characterize compelling and medically relevant membrane protein targets, including the ion channel Slo1-CaV supercomplex, Nan/Wtrw supercomplex, and the full-length tyrosine kinase receptor. We will continuously discuss our results in a structure-property study format to rationalize the roles of detergent structure, host membranes, and membrane proteins in determining membrane protein purification and cryo-EM analysis outcomes. This project will deliver tools that help to better understand biological processes at membrane interfaces and support novel biological findings with no delay.

• DFG Programme: Research Grants