1. The position of the cell penetrating peptide penetratin in SDS micelles determined by NMR
M Lindberg, A Gräslund FEBS Lett. 2001 May 18;497(1):39-44. doi: 10.1016/s0014-5793(01)02433-4.
Penetratin is a 16 residue peptide, RQI KIWFQ NRRMK WKK-amide, with the ability to penetrate cell membranes and a sequence taken from the homeodomain of the Drosophila Antennapedia transcription factor. 600 MHz 1H-nuclear magnetic resonance has been used to study the structure and location of penetratin interacting with a sodium dodecyl sulphate micelle. The positioning of penetratin in the micelle was studied by adding paramagnetic probes (Mn2+ ions, 5-doxyl and 12-doxyl stearic acid) to the solvent. The results show that the peptide is a straight helix positioned with its C-terminus deep inside the micelle and its N-terminus near the surface of the micelle.
2. Membrane interaction and cellular internalization of penetratin peptides
Bart Christiaens, Johan Grooten, Michael Reusens, Alain Joliot, Marc Goethals, Joël Vandekerckhove, Alain Prochiantz, Maryvonne Rosseneu Eur J Biochem. 2004 Mar;271(6):1187-97. doi: 10.1111/j.1432-1033.2004.04022.x.
Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.
3. Tryptophan fluorescence study of the interaction of penetratin peptides with model membranes
Bart Christiaens, et al. Eur J Biochem. 2002 Jun;269(12):2918-26. doi: 10.1046/j.1432-1033.2002.02963.x.
Penetratin is a 16-amino-acid peptide, derived from the homeodomain of antennapedia, a Drosophila transcription factor, which can be used as a vector for the intracellular delivery of peptides or oligonucleotides. To study the relative importance of the Trp residues in the wild-type penetratin peptide (RQIKIWFQNRRMKWKK) two analogues, the W48F (RQIKIFFQNRRMKWKK) and the W56F (RQI KIWFQNRRMKFKK) variant peptides were synthesized. Binding of the three peptide variants to different lipid vesicles was investigated by fluorescence. Intrinsic Trp fluorescence emission showed a decrease in quantum yield and a blue shift of the maximal emission wavelength upon interaction of the peptides with negatively charged phosphatidylserine, while no changes were recorded with neutral phosphatidylcholine vesicles. Upon binding to phosphatidylcholine vesicles containing 20% (w/w) phosphatidylserine the fluorescence blue shift induced by the W56F-penetratin variant was larger than for the W48F-penetratin. Incorporation of cholesterol into the negatively charged lipid bilayer significantly decreased the binding affinity of the peptides. The Trp mean lifetime of the three peptides decreased upon binding to negatively charged phospholipids, and the Trp residues were shielded from acrylamide and iodide quenching. CD measurements indicated that the peptides are random in buffer, and become alpha helical upon association with negatively charged mixed phosphatidylcholine/phosphatidylserine vesicles, but not with phosphatidylcholine vesicles. These data show that wild-type penetratin and the two analogues interact with negatively charged phospholipids, and that this is accompanied by a conformational change from random to alpha helical structure, and a deeper insertion of W48 compared to W56, into the lipid bilayer.
