Abstract
Phosphoinositides play important roles in the regulation of protein recruitment at specialized membrane domains, protein activity, and membrane dynamics. Phosphoinositide–protein interplay occurs via multiple mechanisms and proteins associate with membranes through different binding patterns. Determinations of membrane-binding mode and membrane penetration depth of proteins in lipid bilayer are thus important steps in characterizing the molecular mechanisms of membrane–protein interactions. Here, we show two standard in vitro assays using liposomes, diphenylhexatriene (DPH) anisotropy, and fluorescence quenching by brominated lipids to determine membrane penetration of proteins into lipid bilayer. These methods will provide useful tools to study membrane–protein association and uncover molecular details of protein–lipid interplay, which are important for understanding biological functions of membrane-associated proteins and membrane dynamics.
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Acknowledgments
This work was supported by grants from the Academy of Finland (H.Z.), Jane and Aatos Erkko Foundation (H.Z.), Guangxi distinguished expert funding (H.Z.), FY 2015 Researcher Exchange Program between JSPS and AF (Y.S.), Astellas Foundation for Research on Metabolic Disorders (Y.S.), The Scandinavia-Japan Sasakawa Foundation (Y.S.), The Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care (Y.S.), The Association for Fordays Self-Reliance Support in Japan (Y.S.), The Futaba Research Grant Program of the Futaba Foundation (Y.S.), The NOVARTIS Foundation (Japan) for the Promotion of Science (Y.S.), Okayama Foundation for Science and Technology (Y.S.), Wesco Scientific Promotion Foundation (Y.S.), and JSPS KAKENHI Grant Numbers JP19K23727, JP20K06589 (Y.S.).
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Senju, Y., Zhao, H. (2021). Fluorescence Assays to Study Membrane Penetration of Proteins. In: Botelho, R.J. (eds) Phosphoinositides. Methods in Molecular Biology, vol 2251. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1142-5_16
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DOI: https://doi.org/10.1007/978-1-0716-1142-5_16
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