Membrane potential plays a vital role in the structure and insertion angle of membrane-bound proteins and peptides. Studying these structures under an applied field has previously been technically challenging. Here we present a method by which, using surface-enhanced 2D IR spectroscopy, we can study a membrane-bound polypeptide in a single bilayer. By making these measurements in a spectroelectrical cell with signal enhancement from a rough, gold surface we are able to observed voltage-dependent membrane insertion of a model peptide, alamethicin. Since alamethicin is pH- and voltage- sensitive we can verify the voltage results. The experimental spectra were then modeled with a vibrational excited Hamiltonian, and the insertion angles were able to be extracted by replicating the relative intensities of the 3<sub>10</sub>- and α-helices. Furthermore, the cross peaks between the two helical segments were verified. These results lay the groundwork for further experiments using 2D IR spectroscopy to study the structural impact of applied voltage on membrane proteins and specifically ion channels.

Monitoring voltage-dependent changes of a membrane-bound polypetide using surface-enhanced 2D IR spectroscopy

ACS Fall 2021

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