Otoferlin C2F Domain-Induced Changes in Membrane Structure Observed by Sum Frequency Generation

Abstract

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 orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation 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 phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm À1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. 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 features near 1630 and 1670 cm À1 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 collected SFG spectra, we found the C2F domain of otoferlin orients 22 normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.

Direct Evidence That Mutations within Dysferlin’s C2A Domain Inhibit Lipid Clustering

“…In comparison, for D16A, the time to pressure equilibrium of approximately 22 mN/m (Figure ; trace VI) occurred approximately 20 times faster. It is important to note that the change in surface pressure for lipid monolayer experiments is recorded even if the protein does not dock with the membrane and perform its biological function. , So, the faster increase in surface pressure tells us that the D16A mutant is driven to the lipid interface at a faster rate than the wild type. One possibility is that the D16A variant, substituting a negatively charged aspartic acid to a hydrophobic alanine residue, causes the binding loop to be more hydrophobic, which will increase the protein’s affinity for the interface.…”

“…SFG has previously been used to study lipid monolayers at the air/water interface, − ,− model lipid bilayers, ,,, and small proteins and peptide interaction with each type of model membrane. ,,,− ,− For small proteins and peptides, the direct analysis of SFG amide-I spectra by peak fitting vibrational spectra relating to the CO of the amide backbone can provide information about the orientation and structure. ,, However, since the DYSwt and D16A have complex domain structures, which leads to spectral convolution and interference, it is not possible to obtain unambiguous information about the protein conformation by direct spectral feature identification and fitting. To solve this problem, utilizing the full structural information within the SFG spectra, we have developed a framework for calculating theoretical SFG spectra from protein data bank (PDB) , and molecular dynamics structure files. ,,− We have previously used this method to study the F domain of otoferlin . Thus, spectra for this study were calculated from PDB models.…”

“…37,63,83−87 We have previously used this method to study the F domain of otoferlin. 65 Thus, spectra for this study were calculated from PDB models. Furthermore, by calculating spectra for different protein orientations to the surface normal and subsequently matching experimental and calculated spectra for various different orientations, the surface binding geometry can be determined.…”

Surface analysis tools for characterizing biological materials

“…This includes supplementing SFG experiments with linear vibrational techniques or interpreting complex SFG spectra with the help molecular dynamic simulations. 40 Additionally, the geometry of individual bonds within a larger biomolecule (i.e., individual amino acids within a protein) can be effectively isolated by selective substitution with deuterium-labeling. 36 The resulting collection of previous work includes protocols that demonstrate how SFG-based approaches can be applied to accurately define the geometry of individual domains within a multi domain protein.…”

Membrane Structure of Aquaporin Observed with Combined Experimental and Theoretical Sum Frequency Generation Spectroscopy

“…The challenge to “disentangle” and recover the rich structural information contained in the spectra has been a major challenge in the SFG community for years and has recently led to protocols to calculate theoretical SFG spectra from structure files, which can be used to interpret SFG data of interfacial proteins. − Calculated SFG spectra of proteins and peptides have shown good agreement with experimental SFG spectra. − However, the structural analysis of a protein the size of a membrane protein has not been reported.…”