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Cell signaling is an important process involving complex interactions between lipids and proteins. The myristoylated alanine-rich C-kinase substrate (MARCKS) has been established as a key signaling regulator, serving a range of biological roles. Its effector domain (ED), which anchors the protein to the plasma membrane, induces domain formation in membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS). The mechanisms governing the MARCKS-ED binding to membranes remain elusive. Here, we investigate the composition-dependent affinity and MARCKS-ED-binding-induced changes in interfacial environments using two-dimensional infrared spectroscopy and fluorescence anisotropy. Both negatively charged lipids facilitate the MARCKS-ED binding to lipid vesicles. Although the hydrogen-bonding structure at the lipid-water interface remains comparable across vesicles with varied lipid compositions, the dynamics of interfacial water show divergent patterns due to specific interactions between lipids and peptides. Our findings also reveal that PIP2 becomes sequestered by bound peptides, while the distribution of PS exhibits no discernible change upon peptide binding. Interestingly, PIP2 and PS become colocalized into domains both in the presence and absence of MARCKS-ED. More broadly, this work offers molecular insights into the effects of membrane composition on binding.
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Fluorescence cross-correlation spectroscopy of lipid-peptide interactions on supported lipid bilayers
Electrostatic interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of PIP2 in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptide and the lipid were labeled with orthogonal fluorescent probes, so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Our PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides. Cross-correlation is a direct indication of lipid-peptide binding and complexation. Together with mobility data, we quantified the degree of binding, which offers new insight into this class of lipid-peptide interactions. Overall, this is the first report of lipid-peptide cross-correlation by FCCS, and provides a new route to quantifying the interactions between proteins and lipid membranes, a key interface in cell signaling.
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- Award ID(s):
- 1753060
- PAR ID:
- 10095394
- Date Published:
- Journal Name:
- Advances in biomembranes and lipid self-assembly
- Volume:
- 29
- ISSN:
- 2451-9626
- Page Range / eLocation ID:
- 49 - 68
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/bs.abl.2019.01.005
