Biophysical studies of cell-penetrating peptides and of the RNR inhibitor Sml1
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Several short peptides, so called cell-penetrating peptides, have the capability to transport large hydrophilic cargos through the cell membrane. The objective is to use these peptides as drug carriers and thereby enhance the uptake of drugs into cells.
Three different cell-penetrating peptides are characterized in this thesis. Structure and dynamics of transportan when bound to phospholipid bicelles was determined using NMR. The hydrophobic peptide transportan and its deletion analogue Tp10 both bind to lipid head-group region of the membrane as amphipathic α-helices (papers I & II) and they were found to cause leakage in vesicles (paper IV). The membrane disturbing effect is probably part of how these peptides are translocated through the cell membrane, but also an explanation to why these peptides are found to be toxic in vivo. The high degree of toxicity limits their usefulness. We however also found that the membrane disturbing effect was significantly reduced when a large hydrophilic cargo was attached, which indicates that the properties of the whole peptide-cargo complex has to be taken into account (paper IV).
The highly charged cell-penetrating peptide penetratin is not nearly as membrane disturbing as transportan (papers III and IV). Penetratin binds preferably to negatively charged membranes by electrostatic interactions. We used several different techniques to investigate if penetratin could be translocated through membrane model systems. All experiments consistently suggested that penetratin could not be translocated into model systems. It indicates an endocytotic uptake mechanism into cells rather than a direct membrane penetration (paper III). The ribonucleotide reductase inhibitor protein Sml1 was characterized using NMR and CD spectroscopy (paper V). Three different secondary structure elements were found, in agreement with previous NMR studies, but Sml1 does not have a well defined three-dimensional structure in solution. The N-terminus includes an α-helical region between residues 4-14 and we propose that this region interacts with the C-terminal part of the protein in the monomeric form. The N-terminus is also suggested to be a dimerization interface. Dimers are formed at concentrations above 10 µM in solution. The C-terminal region of Sml1 includes an α-helix between residues 61-80 that is crucial for binding and inhibition of RNR.
Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik , 2008. , 60 p.
cell-penetrating peptides, transportan, bicelles, Sml1
Research subject Biophysics
IdentifiersURN: urn:nbn:se:su:diva-7493ISBN: 978-91-7155-622-6OAI: oai:DiVA.org:su-7493DiVA: diva2:198413
2008-06-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Seelig, Joachim, Professor
Gräslund, Astrid, prof.
List of papers