AVS 66th International Symposium & Exhibition
    Biomaterial Interfaces Division Tuesday Sessions
       Session BI+AS-TuA

Paper BI+AS-TuA7
Identifying the Molecular Mechanisms that Mediate Cell Membrane Repair by Sum Frequency Generation Spectroscopy

Tuesday, October 22, 2019, 4:20 pm, Room A120-121

Session: Biomolecules and Biophysics and Interfaces & Flash Session
Presenter: Joe Baio, Oregon State University
Authors: T.W. Golbek, Oregon State University
S.J. Roeters, Aarhus University, Denmark
T. Weidner, Aarhus University, Denmark
C.P. Johnson, Oregon State University
J.E. Baio, Oregon State University
Correspondent: Click to Email
Movement in everyday life places stress on sarcolemma which creates small tears in the muscle cell membrane. Mutations in this multi-domain dysferlin protein render it unable to repair the membrane and this phenomena is related to diseases such as specific forms of muscular dystrophy. Of particular importance is the moment after the release of calcium from tears in the muscle cell membrane, whereby the release of calcium triggers the C2A domain of dysferlin to dock with a lipid vesicle. Mutations mapped to this domain cause loss of binding ability of the C2A domain. This is the first step of muscle cell membrane repair, therefore there is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to a variant. Here we describe a comparison between the wild type dysferlin C2A and a mutation to the conserved aspartic acids on the domains binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction. A model cell membrane was built with phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide I region for wild type and variant contain features near 1642 cm-1, 1663 cm-1, and 1675 cm-1 related to the C2A domains beta-sandwich secondary structure indicates that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the ratio of the CD3 and CD2 symmetric intensities increases by 170% for the wild type and by 120% for the variant. This study demonstrates and highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.