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Location of TEMPO-PC in Lipid Bilayers: Implications for Fluorescence Quenching

Abstract

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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Acknowledgments

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.

Funding

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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Correspondence to Alexander Kyrychenko.

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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

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Keywords

  • Depth-dependent fluorescence quenching
  • TEMPO
  • Molecular dynamics simulation