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Biophysical studies of cell-penetrating peptides and of the RNR inhibitor Sml1
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.
Keyword [en]
cell-penetrating peptides, transportan, bicelles, Sml1
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-7493ISBN: 978-91-7155-622-6 (print)OAI: oai:DiVA.org:su-7493DiVA: diva2:198413
Public defence
2008-06-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-05-22 Created: 2008-04-16 Last updated: 2015-03-11Bibliographically approved
List of papers
1. NMR solution structure and position of transportan in neutral phospholipid bicelles
Open this publication in new window or tab >>NMR solution structure and position of transportan in neutral phospholipid bicelles
2004 In: FEBS Letters, ISSN 0014-5793, Vol. 567, no (2-3), 265-269 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24841 (URN)
Note
Part of urn:nbn:se:su:diva-7493Available from: 2008-05-22 Created: 2008-04-16Bibliographically approved
2. Dynamics of transportan in bicelles is surface charge dependent
Open this publication in new window or tab >>Dynamics of transportan in bicelles is surface charge dependent
2006 In: Journal of Biomolecular NMR, ISSN 0925-2738, Vol. 35, no 2, 137-147 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24842 (URN)
Note
Part of urn:nbn:se:su:diva-7493Available from: 2008-05-22 Created: 2008-04-16Bibliographically approved
3. A critical reassessment of penetratin translocation across lipid membranes
Open this publication in new window or tab >>A critical reassessment of penetratin translocation across lipid membranes
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2005 In: Biophysical Journal, ISSN 0006-3495, Vol. 89, no 4, 2513-2521 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24843 (URN)
Note
Part of urn:nbn:se:su:diva-7493Available from: 2008-05-22 Created: 2008-04-16Bibliographically approved
4. Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo
Open this publication in new window or tab >>Differential membrane perturbation caused by the cell penetrating peptide Tp10 depending on attached cargo
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2007 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 13, 2389-2393 p.Article in journal (Refereed) Published
Abstract [en]

The membrane leakage caused by the cell penetrating peptide Tp10, a variant of transportan, was studied in large unilamellar vesicles with the entrapped fluorophore calcein. The vesicles were composed of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. A significant decrease in membrane leakage was found when the 55 kDa streptavidin protein was attached to Tp10. When a 5.4 kDa peptide nucleic acid molecule was attached, the membrane leakage was comparable to that caused by Tp10 alone. The results suggest that direct membrane effects may cause membrane translocation of Tp10 alone and of smaller complexes, whereas these effects do not contribute for larger cargoes.

Keyword
cell-penetrating peptides, transportan 10, cell membrane perturbation, streptavidin, peptide nucleic acid
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-22319 (URN)10.1016/j.febslet.2007.04.046 (DOI)000247087600002 ()17485081 (PubMedID)
Available from: 2007-06-12 Created: 2007-06-12 Last updated: 2015-04-21Bibliographically approved
5. The intrinsically disordered RNR inhibitor Sml1 is a dynamic and globular dimer
Open this publication in new window or tab >>The intrinsically disordered RNR inhibitor Sml1 is a dynamic and globular dimer
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Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24845 (URN)
Note
Part of urn:nbn:se:su:diva-7493Available from: 2008-05-22 Created: 2008-04-16 Last updated: 2010-01-13Bibliographically approved

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