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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References
van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124
van Meer G, de Kroon AIPM (2011) Lipid map of the mammalian cell. J Cell Sci 124:5–8
Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93:1019–1137
Saarikangas J, Zhao H, Lappalainen P (2010) Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides. Physiol Rev 90:259–289
Zhao H, Michelot A, Koskela EV et al (2013) Membrane-sculpting BAR domains generate stable lipid microdomains. Cell Rep 4:1213–1223
Saarikangas J, Zhao H, Pykäläinen A et al (2009) Molecular mechanisms of membrane deformation by I-BAR domain proteins. Curr Biol 19:95–107
Drin G, Antonny B (2010) Amphipathic helices and membrane curvature. FEBS Lett 584:1840–1847
Madsen KL, Bhatia VK, Gether U, Stamou D (2010) BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature. FEBS Lett 584:1848–1855
Zhao H, Lappalainen P (2012) A simple guide to biochemical approaches for analyzing protein-lipid interactions. Mol Biol Cell 23:2823–2830
Ladokhin AS (1997) Distribution analysis of depth-dependent fluorescence quenching in membranes: a practical guide. Meth Enzymol 278:462–473
Ladokhin AS (1999) Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations. Biophys J 76:946–955
Ladokhin AS (2014) Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age. Biochim Biophys Acta 1838:2289–2295
McIntosh TJ, Holloway PW (1987) Determination of the depth of bromine atoms in bilayers formed from bromolipid probes. Biochemistry 26:1783–1788
Chattopadhyay A, London E (1987) Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26:39–45
Senju Y, Kalimeri M, Koskela EV et al (2017) Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides. Proc Natl Acad Sci U S A 114:E8977–E8986
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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