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


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.

Graphic Abstract

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3


  1. Abrams FS, London E (1993) Extension of the parallax analysis of membrane penetration depth to the polar region of model membranes: use of fluorescence quenching by a spin-label attached to the phospholipid polar headgroup. Biochemistry 32:10826–10831

  2. Cheng C-Y, Song J, Pas J, Meijer LHH, Han S (2015) DMSO induces dehydration near lipid membrane surfaces. Biophys J 109:330–339

  3. Kyrychenko A, Ladokhin AS (2013) Molecular dynamics simulations of depth distribution of spin-labeled phospholipids within lipid bilayer. J Phys Chem B 117:5875–5885

  4. Kyrychenko A, Ladokhin AS (2014) Refining membrane penetration by a combination of steady-state and time-resolved depth-dependent fluorescence quenching. Anal Biochem 446:19–21

  5. Kyrychenko A, Tobias DJ, Ladokhin AS (2013) Validation of depth-dependent fluorescence quenching in membranes by molecular dynamics simulation of tryptophan octyl ester in POPC bilayer. J Phys Chem B 117:4770–4778

  6. Kyrychenko A, Freites JA, He J, Tobias DJ, Wimley WC, Ladokhin AS (2014) Structural plasticity in the topology of the membrane-interacting domain of HIV-1 gp41. Biophys J 106:610–620

  7. Kyrychenko A, Rodnin MV, Ladokhin AS (2015) Calibration of distribution analysis of the depth of membrane penetration using simulations and depth-dependent fluorescence quenching. J Membr Biol 248:583–594

  8. Kyrychenko A, Rodnin MV, Ghatak C, Ladokhin AS (2017) Joint refinement of FRET measurements using spectroscopic and computational tools. Anal Biochem 522:1–9

  9. Kyrychenko A, Lim NM, Vasquez-Montes V, Rodnin MV, Freites JA, Nguyen LP, Tobias DJ, Mobley DL, Ladokhin AS (2018) Refining protein penetration into the lipid bilayer using fluorescence quenching and molecular dynamics simulations: the case of diphtheria toxin translocation domain. J Membr Biol 251:379–391

  10. Ladokhin AS (1997) Distribution analysis of depth-dependent fluorescence quenching in membranes: a practical guide. In: Ludwig Brand MLJ (ed) Methods in Enzymology, Academic Press, New York, pp 462–473

  11. Ladokhin AS (2014) Measuring membrane penetration with depth-dependent fluorescence quenching: distribution analysis is coming of age. Biochim Biophys Acta - Biomembranes 1838:2289–2295

  12. Ladokhin AS, Holloway PW (1995) Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions. Biophys J 69:506–517

  13. Laudadio E, Galeazzi R, Mobbili G, Minnelli C, Barbon A, Bortolus M, Stipa P (2019) Depth distribution of spin-labeled liponitroxides within lipid bilayers: a combined EPR and molecular dynamics approach. ACS Omega 4:5029–5037

  14. Lee Y, Pincus PA, Hyeon C (2016) Effects of dimethyl sulfoxide on surface water near phospholipid bilayers. Biophys J 111:2481–2491

  15. London E, Ladokhin AS (2002) Measuring the depth of amino acid residues in membrane-inserted peptides by fluorescence quenching. In: Simon SA, McIntosh TJ (eds) Peptide-Lipid Interactions, vol 52. Current topics in membranes. Elsevier, Amsterdam, pp 89–115

  16. Schrader AM, Han S (2017) Location of the TEMPO moiety of TEMPO-PC in lipid bilayers. Biophys J 113:966–969

Download references


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.).

Author information

Correspondence to Alexander Kyrychenko.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Research Involving Human Participants and/or Animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation


  • Depth-dependent fluorescence quenching
  • Molecular dynamics simulation