Abstract
Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: ‘Liposome disruption assay to examine lytic properties of biomolecules’ which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).
Keywords: Membrane, Liposome, Lipid, Disruption, Lysis, Carboxyfluorescein, Leakage, Specificity
Background
Proteins and biomolecules with membrane disruption activities, such as pore formation or membrane fusion, may target specific lipids within membranes. Examples of lipid-specific pore-formation include Plasmodium CelTOS that depends on phosphatidic acid for pore formation, and the cholesterol dependent cytolysins (Jimah et al., 2016; Lukoyanova et al., 2016). CelTOS (cell traversal protein for ookinetes and sporozoites) is a malaria parasite protein that disrupts host cell membranes by pore formation to enable the exit of parasites from invaded host cells during cell traversal (Kariu et al., 2006; Jimah et al., 2016). Cholesterol dependent cytolysins are a large class of pore-forming proteins, including virulence factors of gram positive bacteria such as pneumolysin and listeriolysin (Lukoyanova et al., 2016). Identifying the specific lipids targeted informs the mechanism of membrane disruption that underlies biological function and role of proteins and biomolecules.
Materials and Reagents
Note: See the ‘Notes’ section for a list of materials and reagents used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).
Equipment
Note: See the ‘Notes’ section for a list of equipment used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).
Procedure
Screen to identify specific membrane lipid binding by biomolecules of interest
Data analysis
Data analysis: Screen to identify membrane lipids targeted for membrane disruption by biomolecules Note: Please refer to the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ for a detailed description of data analysis. (Jimah et al., 2017).
Notes
Recipes
Acknowledgments
This work was supported by the Burroughs Wellcome Fund (to NHT) and National Institutes of Health (R56 AI080792 to NHT). This protocol was adapted from Jimah et al., 2016.
References
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