The characterization of the behavior of lipid-attached spin probes in a bilayer is of fundamental importance for correct interpretation of the results of both EPR and fluorescence studies of protein-membrane interactions. The knowledge of the immersion depth of TEMPO spin probe attached to lipid headgroup in TEMPO-PC is critical for the determination of the transverse location of fluorescence probes attached to proteins and peptides. The question of bilayer penetration of TEMPO moiety in TEMPO-PC has recently came into prominence in two studies of interfacial solvation (Cheng et al. in Biophys J 109:330–339, 2015; Lee et al. in Biophys J 111:2481–2491, 2016). Here, we re-examine the arguments on TEMPO penetration using the cross-validation of MD simulations and depth-dependent fluorescence-quenching experiments, which confirms that TEMPO in TEMPO-PC penetrates below the level of phosphate groups. The proper analysis of fluorescence quenching requires the use of Tempo position below the level of phosphate groups; and failure to do so will result in substantial systematic errors in determining the penetration of the labeled site on a membrane protein.
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Supported by NIH GM126778 (A.S.L). A.K. acknowledges Grant 0119U002536 of the Ministry of Education and Science of Ukraine. We are grateful to Aron Fenton and Victor Vasquez-Montes for reading and commenting on the manuscript.
This study was funded by National Institutes of Health (GM126778, A.S.L) and the Ministry of Education and Science of Ukraine (0119U002536, A.K.).
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Kyrychenko, A., Ladokhin, A.S. Location of TEMPO-PC in Lipid Bilayers: Implications for Fluorescence Quenching. J Membrane Biol 253, 73–77 (2020) doi:10.1007/s00232-019-00094-1
- Depth-dependent fluorescence quenching
- Molecular dynamics simulation