Protein Folding and Misfolding at Membrane Interfaces under Electrostatic Control: Combining Vibrational Stark Effect, Surface-Enhanced, and Nano-Infrared Spectroscopy

• Applicant: Dr. Jacek Artur Kozuch
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 500707750

Ever since the discovery of the intrinsic disorder in proteins and peptides, it became clear that both structure as well as the lack of structure can be purposefully encoded in the amino acid sequence, and further binding partners are required to induce disorder-to-order transitions. However, this bears a high risk of severe misfolding and can lead to protein aggregates as observed in Alzheimer’s or in Type 2 Diabetes, which are main causes of dementia. In these protein aggregation diseases, the peptides β-amyloid (Aβ) or islet amyloid polypeptide (IAPP) demonstrate enhanced misfolding towards fibrils at membrane interfaces, leading to dysfunction or even cell death. Membrane electrostatics have been suggested to play a particular role in the structural transitions of the peptides towards amyloid formation: depending on the membrane composition, in particular the content of negatively charged lipids, the peptides undergo an extensive polymorphic behavior adapting various α-helical and β-sheet conformations. Interestingly, some of these species form transmembrane α-helical channel structures early on and interfere with the electrostatic properties of the membrane. This raises the question of not only how lipid composition but also how the transmembrane electrostatic potential gradient can guide the trajectory of folding events of these polymorphic, disease-relevant peptides. To provide insight into this question, I propose to utilize an experimental strategy centered around the advanced infrared (IR) spectroscopic methods of surface-enhanced IR absorption (SEIRA) and nanoscale IR spectroscopy in liaison with tethered bilayer lipid membrane systems (tBLMs) and the vibrational Stark effect (VSE). The combination of tBLMs and SEIRA will enable to monitor the folding processes of Aβ and IAPP in-situ at and in membranes of various compositions and under the direct control of the transmembrane potential. Furthermore, using tools from molecular biology, we will introduce CN group-containing non-natural amino acids into Aβ and IAPP, which will enable to quantify changes in local electrostatic environments using the framework of the VSE. The latter will also provide a strategy to identify if different polymorphic β-sheet amyloid species have formed, which otherwise can be difficult to discern using IR spectroscopy. Lastly, we will extend the SEIRA-tBLM approach towards nanoscale IR spectroscopic methods, which utilize atomic force microscopy tips as antennas to detect IR spectra of heterogeneous systems below the diffraction limit, that is with a spatial resolution of ca. 30 nm. The results of this project will contribute to the understanding of how specific electrostatic conditions at lipid membranes can steer polymorphic proteins along their flat conformational landscape towards folded or misfolded structures in the context of amyloid diseases.

• DFG Programme: Research Grants