Vibrational probe groups could provide a unique site-specific method to display the dynamic structures of membrane proteins. This work focuses on three “vibrational probe” functional groups with unique spectral signals: the azido, thiocyanate, and alkyne probe groups. In order to build depth-dependency profiles of these groups in a bilayer membrane environment, a 23-amino acid symmetric polyleucine transmembrane helical peptide is used as a model system. Synthetic peptides with azide groups substituted at L12, L9, L7, and L5 (starting from the center of the peptide sequence and moving out towards the head groups) were purified via a non-canonical reversed-phase HPLC protocol and then integrated into POPC membranes. Both rehydrated bilayer film samples and vesicle samples were made with probe-labeled peptides. Far-UV circular dichroism (CD) was utilized to see whether the peptides were helical and vertically inserted into the POPC membranes. Infrared spectroscopy (IR) was used to study the thiocyanate and azido probes’ behaviors in different solvent environments, and the results in each case cannot be explained by simple solvatochromism of either probe. Raman spectra of alkyne groups at the same peptide positions exhibited site-specific changes but did not show a strong depth-dependency.<br/>The complete depth-dependency profiles for each of these probe groups will serve as a spectroscopic “rulers” and provide guidelines for the future use of these probes in membrane proteins. We have also run molecular dynamics simulations of the model peptides in a simulated POPC bilayer environment, and several observables from MD provide possible physical explanations for the experimental results for each probe group. By directly linking experiments and simulations in this model peptide system, we will provide a fully characterized “spectroscopic ruler” for understanding vibrational probe behavior in any membrane protein. With information from this work, different probe groups could be chosen and incorporated into both peripheral and intrinsic membrane proteins.

Building a spectroscopic inventory of membrane depth-dependent characteristics of vibrational probe groups using model transmembrane peptides via experiments and simulations

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.