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Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching

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Abstract

Determination of the depth of membrane penetration provides important information for studies of membrane protein folding and protein–lipid interactions. Here, we use a combination of molecular dynamics (MD) simulations and depth-dependent fluorescence quenching to calibrate the methodology for extracting quantitative information on membrane penetration. In order to investigate the immersion depth of the fluorescent label in lipid bilayer, we studied 7-nitrobenz-2-oxa-1,3-diazole (NBD) attached to the lipid headgroup in NBD-PE incorporated into POPC bilayer. The immersion depth of NBD was estimated by measuring steady-state and time-resolved fluorescence quenching with spin-labeled lipids co-incorporated into lipid vesicles. Six different spin-labeled lipids were utilized: one with headgroup-attached Tempo probe (Tempo-PC) and five with acyl chain-labeled n-Doxyl moieties (n-Doxyl-PC where n is a chain labeling position equal to 5, 7, 10, 12, and 14, respectively). The Stern–Volmer analysis revealed that NBD quenching in membranes occurs by both static and dynamic collisional quenching processes. Using the methodology of Distribution Analysis, the immersion depth and the apparent half-width of the transversal distributions of the NBD moiety were estimated to be 14.7 and 6.7 Å, respectively, from the bilayer center. This position is independently validated by atomistic MD simulations of NBD-PE lipids in a POPC bilayer (14.4 Å). In addition, we demonstrate that MD simulations of the transverse overlap integrals between dye and quencher distributions can be used for proper analysis of the depth-dependent quenching profile. Finally, we illustrate the application of this methodology by determining membrane penetration of site selectively labeled mutants of diphtheria toxin T-domain.

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Abbreviations

NBD-PE:

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)

Tempo-PC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho(TEMPO)choline

n-Doxyl-PC:

1-Palmitoyl-2-stearoyl-(n-Doxyl)-sn-glycero-3-phosphocholine

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

LUV:

Large unilamellar lipid vesicle

FRET:

Förster resonance energy transfer

MD simulation:

Molecular dynamics simulation

DA:

Distribution analysis

COM:

Center of mass

References

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Armstrong VT, Brzustowicz MR, Wassall SR, Jenski LJ, Stillwell W (2003) Rapid flip-flop in polyunsaturated (docosahexaenoate) phospholipid membranes. Arch Biochem Biophys 414:74–82

    Article  CAS  PubMed  Google Scholar 

  • Bartlett GR (1959) Phosphorus assay in column chromatography. J Biol Chem 234:466–468

    CAS  PubMed  Google Scholar 

  • Bennett MJ, Eisenberg D (1994) Refined structure of monomelic diphtheria toxin at 2.3 Å resolution. Protein Sci 3:1464–1475

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690

    Article  CAS  Google Scholar 

  • Berger O, Edholm O, Jähnig F (1997) Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys J 72:2002–2013

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chattopadhyay A, London E (1987) Parallax method for direct measurement of membrane penetration depth utilizing fluorescence quenching by spin-labeled phospholipids. Biochemistry 26:39–45

    Article  CAS  PubMed  Google Scholar 

  • Chattopadhyay A, London E (1988) Spectroscopic and ionization properties of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeledlipidsinmodelmembranes. Biochim Biophys Acta 938:24–34

    Article  CAS  PubMed  Google Scholar 

  • Chattopadhyay A, Mukherjee S (1999a) Depth-dependent solvent relaxation in membranes: wavelength-selective fluorescence as a membrane dipstick. Langmuir 15:2142–2148

    Article  CAS  Google Scholar 

  • Chattopadhyay A, Mukherjee S (1999b) Red edge excitation shift of a deeply embedded membrane probe: implications in water penetration in the bilayer. J Phys Chem B 103:8180–8185

    Article  CAS  Google Scholar 

  • Darden T, York D, Pedersen L (1993) Particle mesh Ewald: an N × log(N) method for Ewald sums in large systems. J Chem Phys 98:10089–10092

    Article  CAS  Google Scholar 

  • Feigenson GW (1997) Partitioning of a fluorescent phospholipid between fluid bilayers: dependence on host lipid acyl chains. Biophys J 73:3112–3121

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Filipe HAL, Moreno MJ, Loura LMS (2011) Interaction of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled fatty amines with 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphocholine bilayers: a molecular dynamics study. J Phys Chem B 115:10109–10119

    Article  CAS  PubMed  Google Scholar 

  • Greiner AJ, Pillman HA, Worden RM, Blanchard GJ, Ofoli RY (2009) Effect of hydrogen bonding on the rotational and translational dynamics of a headgroup-bound chromophore in bilayer lipid membranes. J Phys Chem B 113:13263–13268

    Article  CAS  PubMed  Google Scholar 

  • Haldar S, Kombrabail M, Krishnamoorthy G, Chattopadhyay A (2012) Depth-dependent heterogeneity in membranes by fluorescence lifetime distribution analysis. J Phys Chem Lett 3:2676–2681

    Article  CAS  PubMed  Google Scholar 

  • Hermans J, Berendsen HJC, Van Gunsteren WF, Postma JPM (1984) A consistent empirical potential for water–protein interactions. Biopolymers 23:1513–1518

    Article  CAS  Google Scholar 

  • Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472

    Article  CAS  Google Scholar 

  • Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38

    Article  CAS  PubMed  Google Scholar 

  • Huster D, Müller P, Arnold K, Herrmann A (2003) Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy. Eur Biophys J 32:47–54

    CAS  PubMed  Google Scholar 

  • Kaback HR, Wu J (1999) What to do while awaiting crystals of a membrane transport protein and thereafter. Acc Chem Res 32:805–813

    Article  CAS  Google Scholar 

  • Kachel K, Asuncion-Punzalan E, London E (1998) The location of fluorescence probes with charged groups in model membranes. Biochim Biophys Acta 1374:63–76

    Article  CAS  PubMed  Google Scholar 

  • Kyrychenko A (2010) A molecular dynamics model of rhodamine-labeled phospholipid incorporated into a lipid bilayer. Chem Phys Lett 485:95–99

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kyrychenko A, Posokhov YO, Rodnin MV, Ladokhin AS (2009) Kinetic intermediate reveals staggered pH-dependent transitions along the membrane insertion pathway of the diphtheria toxin T-domain. Biochemistry 48:7584–7594

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kyrychenko A, Wu F, Thummel RP, Waluk J, Ladokhin AS (2010) Partitioning and localization of environment-sensitive 2-(2′-pyridyl)- and 2-(2′-pyrimidyl)-indoles in lipid membranes: a joint refinement using fluorescence measurements and molecular dynamics simulations. J Phys Chem B 114:13574–13584

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Kyrychenko A, Sevriukov IY, Syzova ZA, Ladokhin AS, Doroshenko AO (2011) Partitioning of 2,6-bis(1H-benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations. Biophys Chem 154:8–17

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Ladokhin AS (1997) Distribution analysis of depth-dependent fluorescence quenching in membranes: a practical guide. In: Ludwig Brand MLJ (ed) Methods in Enzymology, vol 278. Academic Press, New York, pp 462–473

    Google Scholar 

  • Ladokhin AS (1999a) Analysis of protein and peptide penetration into membranes by depth-dependent fluorescence quenching: theoretical considerations. Biophys J 76:946–955

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ladokhin AS (1999b) Evaluation of lipid exposure of tryptophan residues in membrane peptides and proteins. Anal Biochem 276:65–71

    Article  CAS  PubMed  Google Scholar 

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

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ladokhin AS, Legmann R, Collier RJ, White SH (2004) Reversible refolding of the diphtheria toxin T-domain on lipid membranes. Biochemistry 43:7451–7458

    Article  CAS  PubMed  Google Scholar 

  • London E, Ladokhin AS (2002) Measuring the depth of amino acid residues in membrane-inserted peptides by fluorescence quenching. Curr Top Membr 52:89–115

    Article  CAS  Google Scholar 

  • Loura LMS (2012) Lateral distribution of NBD-PC fluorescent lipid analogs in membranes probed by molecular dynamics-assisted analysis of Förster Resonance Energy Transfer (FRET) and fluorescence quenching. Int J Mol Sci 13:14545–14564

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Loura LMS, Prates Ramalho JP (2011) Recent developments in molecular dynamics simulations of fluorescent membrane probes. Molecules 16:5437–5452

    Article  CAS  PubMed  Google Scholar 

  • Loura LMS, Ramalho JPP (2007) Location and dynamics of acyl chain NBD-labeled phosphatidylcholine (NBD-PC) in DPPC bilayers. A molecular dynamics and time-resolved fluorescence anisotropy study. Biochim Biophys Acta 1768:467–478

    Article  CAS  PubMed  Google Scholar 

  • Loura LMS, Fernandes F, Fernandes AC, Ramalho JPP (2008) Effects of fluorescent probe NBD-PC on the structure, dynamics and phase transition of DPPC. A molecular dynamics and differential scanning calorimetry study. Biochim Biophys Acta 1778:491–501

    Article  CAS  PubMed  Google Scholar 

  • Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161–168

    Article  CAS  PubMed  Google Scholar 

  • Mazères S, Schram V, Tocanne JF, Lopez A (1996) 7-nitrobenz-2-oxa-1,3-diazole-4-yl-labeled phospholipids in lipid membranes: differences in fluorescence behavior. Biophys J 71:327–335

    Article  PubMed Central  PubMed  Google Scholar 

  • Mukherjee S, Raghuraman H, Dasgupta S, Chattopadhyay A (2004) Organization and dynamics of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: a fluorescence approach. Chem Phys Lipids 127:91–101

    Article  CAS  PubMed  Google Scholar 

  • Posokhov YO, Ladokhin AS (2006) Lifetime fluorescence method for determining membrane topology of proteins. Anal Biochem 348:87–93

    Article  CAS  PubMed  Google Scholar 

  • Pucadyil TJ, Mukherjee S, Chattopadhyay A (2007) Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching. J Phys Chem B 111:1975–1983

    Article  CAS  PubMed  Google Scholar 

  • Rodnin MV, Posokhov YO, Contino-Pepin C, Brettmann J, Kyrychenko A, Palchevskyy SS, Pucci B, Ladokhin AS (2008) Interactions of fluorinated Surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins. Biophys J 94:4348–4357

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rodnin MV, Kyrychenko A, Kienker P, Sharma O, Posokhov YO, Collier RJ, Finkelstein A, Ladokhin AS (2010) Conformational switching of the diphtheria toxin T domain. J Mol Biol 402:1–7

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sassaroli M, Ruonala M, Virtanen J, Vauhkonen M, Somerharju P (1995) Transversal distribution of acyl-linked pyrene moieties in liquid-crystalline phosphatidylcholine bilayers. A fluorescence quenching study. Biochemistry 34:8843–8851

    Article  CAS  PubMed  Google Scholar 

  • Shrive JDA, Kanagalingam S, Krull UJ (1996) Influence of structural heterogeneity on the fluorimetric response characteristics of lipid membranes containing nitrobenzoxadiazolyl-dipalmitoyl-phosphatidylethanolamine. Langmuir 12:4921–4928

    Article  CAS  Google Scholar 

  • Skaug MJ, Longo ML, Faller R (2009) Computational studies of Texas Red-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-model building and applications. J Phys Chem B 113:8758–8766

    Article  CAS  PubMed  Google Scholar 

  • Stubbs CD, Williams BW, Boni LT, Hoek JB, Taraschi TF, Rubin E (1989) On the use of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethnolamine in the study of lipid polymorphism. Biochim Biophys Acta 986:89–96

    Article  CAS  PubMed  Google Scholar 

  • Vaish V, Sanyal S (2011) Non steroidal anti-inflammatory drugs modulate the physicochemical properties of plasma membrane in experimental colorectal cancer: a fluorescence spectroscopic study. Mol Cell Biochem 358:161–171

    Article  CAS  PubMed  Google Scholar 

  • Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJC (2005) GROMACS: fast, flexible, and free. J Comput Chem 26:1701–1718

    Article  Google Scholar 

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Acknowledgments

This work was performed using computational facilities of the joint computational cluster of SSI “Institute for Single Crystals” and Institute for Scintillation Materials of National Academy of Science of Ukraine incorporated into Ukrainian National Grid. This research was supported by National Institutes of Health Grant GM-069783 (A.S.L.). A.K. also acknowledges support of Grant 0113U002426 of Ministry of Education and Science of Ukraine.

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Authors and Affiliations

  1. Institute of Chemistry and School of Chemistry, V. N. Karazin Kharkiv National University, 4 Svobody Square, Kharkiv, 61022, Ukraine

    Alexander Kyrychenko

  2. Department of Biochemistry and Molecular Biology, Kansas University Medical Center, Kansas City, KS, 66160-7421, USA

    Alexander Kyrychenko, Mykola V. Rodnin & Alexey S. Ladokhin

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  1. Alexander Kyrychenko
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  2. Mykola V. Rodnin
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  3. Alexey S. Ladokhin
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Correspondence to Alexander Kyrychenko.

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Kyrychenko, A., Rodnin, M.V. & Ladokhin, A.S. Calibration of Distribution Analysis of the Depth of Membrane Penetration Using Simulations and Depth-Dependent Fluorescence Quenching. J Membrane Biol 248, 583–594 (2015). https://doi.org/10.1007/s00232-014-9709-1

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