A simple fluorescence-spectroscopic membrane translocation assay.
We have established a combination of fluorescence-spectroscopic uptake, release, and dilution experiments as a powerful tool for studying the translocation of fluorescent compounds across lipid membranes, demonstrating this through intrinsic tryptophan fluorescence for the interaction of the cell-penetrating peptide penetratin with phospholipid membranes, for which conflicting results have been reported. We found that penetratin is not membrane-permeant under the conditions used here. To confirm this finding and to validate the approach, we also employed an established titration-calorimetric method, the results of which were in excellent agreement with a thermodynamic analysis of the fluorescence-spectroscopic experiments. Further support was provided by a comparison with published data obtained under similar conditions by using a variety of techniques. Unlike these methods, however, the new approach allows consistent and simultaneous assessment of membrane binding and transbilayer movement without depending on extrinsic labels attached to the molecule of interest or on reporter moieties inserted into the lipid membrane.
Keller
Sandro
S
Leibniz Institute of Molecular Pharmacology FMP, Robert-Rössle-Strasse 10, 13125 Berlin, Germany. keller@fmp-berlin.de
Böthe
Matthias
M
Bienert
Michael
M
Dathe
Margitta
M
Blume
Alfred
A
D001671:Biochemistry / Q000379:methods; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D057846:Cell-Penetrating Peptides; D008051:Lipid Bilayers / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015233:Models, Statistical; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D021381:Protein Transport; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry*
D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D008051:Lipid Bilayers; D008055:Lipids; D010455:Peptides; D010743:Phospholipids; D014364:Tryptophan; C414409:penetratin