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bioRxiv · 10.1101/2025.08.29.672991

Development of an EPR-based methodology to study protein-lipid interaction

Piersson, C.·Prakash, S.·Lublin, V.·Rossotti, M.·Fischer, B.·Srivastava, M.·Fichou, Y.
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Abstract

The interaction of protein with other biomolecules is central to all cellular processes. In particular, protein-lipid interactions play an essential role in regulating soluble and membrane protein function, structure, and dynamics. However, probing these interactions remains challenging due to the complexity and heterogeneity of membranes. Various methods have been developed to characterize protein-membrane interaction, each presenting advantages and limitations. This study presents a robust methodology based on continuous-wave Electron Paramagnetic Resonance (CW-EPR) spectroscopy to characterize protein-membrane interactions. We focused on the protein Tau, an intrinsically disordered protein associated with neurodegenerative diseases. We show that the interaction of labelled Tau with lipids gives rise to a very distinct lineshape, which can be used to quantify the fraction of bound protein. This allows to obtain the apparent binding mode and affinity through titration experiments. In addition, we show that a single measurement provides the absolute concentration of free and bound protein. We argue that this information, which is rarely obtained by other methods providing relative signals, is very useful for mechanistic studies. Furthermore, using mathematical modeling, we developed a minimal-data approach and demonstrated that a single EPR measurement can be used to derive binding constants. The approach is applied to the Taumembrane interaction occurring in different conditions affecting the binding behavior. The presented methodology is expected to be applicable to other proteins. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/672991v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@19cf0c5org.highwire.dtl.DTLVardef@833209org.highwire.dtl.DTLVardef@149fd8corg.highwire.dtl.DTLVardef@a4f50a_HPS_FORMAT_FIGEXP M_FIG C_FIG TOC Figure. Schematic representation of EPR-based analysis of Tau-membrane interactions using spin-labeled Tau and POPS vesicles

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Piersson, C., Prakash, S., Lublin, V., Rossotti, M., Fischer, B., Srivastava, M., Fichou, Y.. 2025-09-02. Development of an EPR-based methodology to study protein-lipid interaction. https://doi.org/10.1101/2025.08.29.672991

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