| AVS 65th International Symposium & Exhibition | |
| Biomaterial Interfaces Division | Monday Sessions |
| Session BI+AS+IPF+MN-MoA |
| Session: | Advanced Imaging and Structure Determination of Biomaterials Research |
| Presenter: | Joe Baio, Oregon State University |
| Authors: | T.W. Golbek, Oregon State University T. Weidner, Aarhus University, Denmark C.P. Johnson, Oregon State University J.E. Baio, Oregon State University |
| Correspondent: | Click to Email |
Proteins that contain C2 domains are involved in a variety of biological processes including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein’s lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here we describe the first in situ probe of the protein structure of otoferlin’s C2F domain interacting with a cell membrane surface. To identify this protein’s structure at the lipid interface we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphoserine (PS) and phosphocholine (PC). SFG studies that examined the ordering of the lipids that make up the model membrane, demonstrate that lipid fusion occurs after docking of the otoferlin C2F domain via the observation of a 62% increase in amplitude from the SFG signal near 2075 cm-1 assigned to specific groups within the model membrane. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide I region contain peaks near 1621 cm-1 and 1672 cm-1 related to the C2F domains beta-sandwich secondary structure, thus, indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collect SFG spectra, we found the C2F domain of otoferlin orients 32° normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane. Furthermore, we show first experimental view of any C2 domain of otoferlin docked at the membrane interface, thereby, validating SFG as a method to probe C2 domain-membrane interfaces.