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bioRxiv · 10.64898/2026.07.27.740883

Analytical methodology for unveiling the physical effect of detergents on the function of full-length membrane proteins using a single-molecule tracking system

Hoshi, H.·Kawano, K.·Fujiwara, T.·Yamaoka, Y.·Kuroda, Y.·Kurokawa, K.·Taniguchi, A.·Nagase, K.·Takasu, K.·Matsuzaki, K.
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Abstract

G protein-coupled receptors (GPCRs) are important targets for drug discovery because they are the largest and most diverse family of membrane proteins in the human body. As exemplified by {beta}2-adrenergic receptor ({beta}2AR), which is a typical class-A GPCRs, they are prone to denaturation and inactivation after solubilization. Although diverse techniques have been developed, current structural and functional analyses are still limited to proteins with relatively stable and high expression. However, some mutant variants and misfolded proteins involved in diseases have extremely low expression levels and may be difficult to analyze. To overcome these limitations, we established a novel analytical platform for evaluating the ligand-binding ability of full-length {beta}2AR as a model at the single-molecule level without purification and addressing challenges such as membrane proteins with low expression levels and structural instability. This method enables the direct use of unpurified receptors immediately after solubilization and allows us to use only a small amount of sample ([~]10 ng) for observation and to distinguish between specific and nonspecific ligand binding by fitting. Furthermore, we unveiled the physical properties of detergents on the structural stability of solubilized receptors and found that the lateral pressure within detergent micelles affects ligand-binding ability. Detergents that provided a fluid microenvironment were able to maintain ligand-binding ability for several days even after solubilization; conversely, detergents that provided a rigid microenvironment caused the protein to lose its activity earlier. Our method could be a promising tool for the structural and functional analysis of membrane proteins untargeted until now. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/740883v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@3d274corg.highwire.dtl.DTLVardef@defd0forg.highwire.dtl.DTLVardef@1b81eb4org.highwire.dtl.DTLVardef@e7c275_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Hoshi, H., Kawano, K., Fujiwara, T., Yamaoka, Y., Kuroda, Y., Kurokawa, K., Taniguchi, A., Nagase, K., Takasu, K., Matsuzaki, K.. 2026-07-30. Analytical methodology for unveiling the physical effect of detergents on the function of full-length membrane proteins using a single-molecule tracking system. https://doi.org/10.64898/2026.07.27.740883

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