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

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Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. 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 that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain 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 with a model cell membrane composed of 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 the wild type and variant contain features near 1642, 1663, and 1675 cm-1 related to the C2A domain β-sandwich secondary structure, indicating 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 increase in the ratio of the CD3 and CD2 symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.

OriginalsprogEngelsk
TidsskriftJournal of Physical Chemistry B
Vol/bind125
Nummer1
Sider (fra-til)148-157
Antal sider10
ISSN1520-6106
DOI
StatusUdgivet - jan. 2021

Bibliografisk note

Funding Information:
This work was supported by John C. Erkkila, M.D., Endowment for Health and Human Performance, NIH National Institute of Deafness and Other Communication Disorders (NIDCD) Grant R01DC014588 to C.P.J. and the National Science Foundation award #1905091. T.W.G. would like to thank the Lundbeck Foundation (postdoc grant R322-2019-2461). S.J.R. acknowledges the Lundbeck Foundation for funding through fellowship grant R303-2018-349.

Publisher Copyright:
© 2020 American Chemical Society.

Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.

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