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

Area of Science:

• Biophysics
• Molecular Biology
• Neuroscience

Background:

• C2 domain proteins are vital for cellular functions like signaling and membrane repair.
• Mutations in otoferlin's C2F domain disrupt lipid binding, leading to hearing loss.
• Understanding otoferlin's membrane interaction is critical for addressing hearing impairment.

Purpose of the Study:

• To determine the orientation of otoferlin's C2F domain at the lipid membrane interface.
• To investigate the structural changes induced by otoferlin binding to model cell membranes.

Main Methods:

• Utilized sum frequency generation (SFG) vibrational spectroscopy.
• Coupled experimental SFG with simulated SFG spectra for quantitative analysis.
• Constructed a model cell membrane using phosphatidylserine and phosphatidylcholine.

Main Results:

• Observed a 62% increase in SFG signal amplitude near 2075 cm⁻¹, indicating lipid reorientation and potential membrane curvature.
• Identified spectral features in the amide-I region (1630-1670 cm⁻¹) confirming the C2F domain's beta-sandwich structure and specific binding orientation.
• Quantified the C2F domain's orientation at 22° normal to the lipid surface.

Conclusions:

• The otoferlin C2F domain docks to lipid membranes in a defined orientation.
• This orientation influences lipid ordering and membrane structure.
• Provides a structural basis for understanding otoferlin's role in hearing and the impact of mutations.