Abstract
Assessing the ability of biomolecules or drugs to overcome lipid membranes in a receptor-independent way is of great importance in both basic research and applications involving the use of liposomes. A combination of uptake, release, and dilution experiments performed by steady-state fluorescence spectroscopy provides a powerful, straightforward, and inexpensive way of monitoring membrane translocation of fluorescent compounds. This is particularly true for peptides and proteins carrying intrinsic tryptophan residues, which eliminates the need for attaching extrinsic labeling moieties to the compound of interest. The approach encompasses three different kinds of fluorescence titrations and some simple calculations that can be carried out in a spreadsheet program. A complete set of experiments and data analyses can typically be completed within two days.
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References
Heerklotz HH, Binder H, Epand RM (1999) A “release” protocol for isothermal titration calorimetry. Biophys J 76:2606-2613
Heerklotz H, Seelig J (2000) Titration calorimetry of surfactant-membrane partitioning and membrane solubilization. Biochim Biophys Acta 1508:69-85
Heerklotz H (2004) The microcalorimetry of lipid membranes. J Phys Condens Matter 16:R441-R467
Hagen V, Dekowski B, Nache V, Schmidt R, Geissler D, Lorenz D, Eichhorst J, Keller S, Kaneko H, Benndorf K, Wiesner B (2005) Coumarinylmethyl esters for ultrafast release of high concentrations of cyclic nucleotides upon one- and two-photon photolysis. Angew Chem Int Ed 44:7887-7891
Cambridge SB, Geissler D, Keller S, Cürten B (2006) A caged doxycycline analogue for photoactivated gene expression. Angew Chem Int Ed 45:2229-2231
Gilbert D, Funk K, Dekowski B, Lechler R, Keller S, Möhrlen F, Frings S, Hagen V (2007) Caged capsaicins: new tools for the examination of TRPV1 channels in somatosensory neurons. ChemBioChem 8:89-97
Heerklotz H, Szadkowska H, Anderson T, Seelig J (2003) The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton. J Mol Biol 329:793-799
Tsamaloukas A, Szadkowska H, Slotte PJ, Heerklotz H (2005) Interactions of cholesterol with lipid membranes and cyclodextrin characterized by calorimetry. Biophys J 89:1109-1119
Tsamaloukas A, Szadkowska H, Heerklotz H (2006) Thermodynamic comparison of the interactions of cholesterol with unsaturated phospholipid and sphingomyelins. Biophys J 90:4479-4487
Keller S, Heerklotz H, Blume A (2006) Monitoring lipid membrane translocation of sodium dodecyl sulfate by isothermal titration calorimetry. J Am Chem Soc 128:1279-1286
Keller S, Böthe M, Bienert M, Dathe M, Blume A (2007) A simple fluorescence-spectroscopic membrane translocation assay. ChemBioChem 8:546-552
Tsamaloukas AD, Keller S, Heerklotz H (2007) Uptake and release protocol for assessing membrane binding and permeation by way of isothermal titration calorimetry. Nat Protoc 2:695-704
Bárány-Wallje E, Keller S, Serowy S, Geibel S, Pohl P, Bienert M, Dathe M (2005) A critical reassessment of penetratin translocation across lipid membranes. Biophys J 89:2513-2521
Derossi D, Chassaing G, Prochiantz A (1998) Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 8:84-87
Drin G, Déméné H, Temsamani J, Brasseur R (2001) Translocation of the pAntp peptide and its amphipathic analogue AP-2AL. Biochemistry 40:1824-1834
Persson D, Thorén PEG, Esbjörner EK, Goksör M, Lincoln P, Nordén B (2004) Vesicle size-dependent translocation of penetratin analogs across lipid membranes. Biochim Biophys Acta 1665:142-155
Thorén PEG, Persson D, Karlsson M, Nordén B (2000) The Antennapedia peptide penetratin translocates across lipid bilayers - the first direct observation. FEBS Lett 482:265-268
Terrone D, Sang SLW, Roudaia L, Silvius JR (2003) Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential. Biochemistry 42:13787-13799
Chico DE, Given RL, Miller BT (2003) Binding of cationic cell-permeable peptides to plastic and glass. Peptides 24:3-9
Persson D, Thorén PEG, Herner M, Lincoln P, Nordén B (2003) Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes. Biochemistry 42:421-429
Hope MJ, Bally MB, Webb G, Cullis PR (1985) Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim Biophys Acta 812:55-65
Mayer LD, Hope MJ, Cullis PR (1986) Vesicles of variable sizes produced by a rapid extrusion procedure. Biochim Biophys Acta 858:161-168
MacDonald RC, MacDonald RI, Menco BPM, Takeshita K, Subbarao NK, Hu LR (1991) Small-volume extrusion apparatus for preparation of large, unilamellar vesicles. Biochim Biophys Acta 1061:297-303
Ladokhin AS, Jayasinghe S, White SH (2000) How to measure and analyze tryptophan fluorescence in membranes properly, and why bother? Anal Biochem 285:235-245
Chatterjee S, Banerjee DK (2002) Preparation, isolation, and characterization of liposomes containing natural and synthetic lipids. Methods Mol Biol 199:3-16
Acknowledgments
We thank Heike Nikolenko (FMP) and Matthias Böthe (Robert Koch Institute, Berlin, Germany) for excellent technical assistance and Sebastian Fiedler (FMP) for helpful comments on the manuscript. We are indebted to Dr. Michael Beyermann, Dagmar Krause, and Bernhard Schmikale for synthesis and purification and to Drs. Eberhard Krause and Michael Schümann (all FMP) for mass-spectrometric characterization of penetratin peptide. This work was supported by the European Commission with grant No. QLK3-CT-2002-01989 to S.K.
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Broecker, J., Keller, S. (2010). Membrane Translocation Assayed by Fluorescence Spectroscopy. In: Weissig, V. (eds) Liposomes. Methods in Molecular Biology™, vol 606. Humana Press. https://doi.org/10.1007/978-1-60761-447-0_19
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DOI: https://doi.org/10.1007/978-1-60761-447-0_19
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