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Biophysical studies of peptides with functions in biotechnology and biology
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

My thesis concerns spectroscopic studies (NMR, CD and fluorescence) of peptides with functions in biotechnology and biology, and their interactions with a model membrane (large unilamellar phospholipid vesicles).

The resorufin-based arsenical hairpin binder (ReAsH) bound to a short peptide is a useful fluorescent tag for genetic labeling of proteins in living cells. A hairpin structure with some resemblance to type II β-turn was determined by NMR structure calculations (Paper I).

Cell-penetrating peptides (CPPs) are short (30-35 residues), often rich in basic amino acids such as Arg. They can pass through the cell membrane and deliver bioactive cargoes, making them useful for biotechnical and pharmacological applications. The mechanisms of cellular uptake and membrane translocation are under debate. Understanding the mechanistic aspects of CPPs is the major focus of Papers II, III, and IV.

The effect of the pyrenebutyrate (PB) on the cellular uptake, membrane translocation and perturbation of several CPPs from different subgroups was investigated (Paper II). We concluded that both charge and hydrophobicity of the CPP affect the cellular uptake and membrane translocation efficiency.

Endosomal escape is a crucial challenge for the CPP applications. We modeled the endosome and endosomal escape for different CPPs to investigate the corresponding molecular mechanisms (Papers III and IV). Hydrophobic CPPs were able to translocate across the model membrane in the presence of a pH gradient, produced by bacteriorhodopsin proton pumping, whereas a smaller effect was observed for hydrophilic CPPs.

Dynorphin A (Dyn A) peptide mutations are associated with neurodegenerative disorders, without involvement of the opioid receptors. The non-opioid activities of Dyn A may involve membrane perturbations. Model membrane-perturbations by three Dyn A mutants were investigated (Paper V). The results showed effects to different degrees largely in accordance with their neurotoxic effects.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2012. , 75 p.
Keyword [en]
Genetic fluorescence label, Biarsenical tetracysteine motif, Cell-penetrating peptides, Large unilamellar vesicles, Pyrenebutyrate, Endosomal escape, Membrane perturbation, Bacteriorhodopsin, Dynorphin
National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-66948ISBN: 978-91-7447-417-6 (print)OAI: oai:DiVA.org:su-66948DiVA: diva2:469010
Public defence
2012-02-14, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2012-01-23 Created: 2011-12-22 Last updated: 2013-04-09Bibliographically approved
List of papers
1. Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy
Open this publication in new window or tab >>Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy
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2009 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 131, no 13, 4613-4615 p.Article in journal (Refereed) Published
Abstract [en]

The biarsenical−tetracysteine motif is a useful tag for genetic labeling of proteins with small molecules in living cells. The present study concerns the structure of a 12 amino acid peptide FLNCCPGCCMEP bound to the fluorophore ReAsH based on resorufin. 1H NMR spectroscopy was used to determine the solution structure of the complex formed between the peptide and the ReAsH moiety. Structure calculations based on the NMR results showed that the backbone structure of the peptide is fairly well defined, with a hairpinlike turn, similar to a type-II β-turn, formed by the central CPGC segment. The most stable complex was formed when As2 was bonded to C4 and C5 and As1 to C8 and C9. Two clear NOESY cross-peaks between the Phe1 side chain and ReAsH confirmed the close positioning of the phenyl ring of Phe1 and ReAsH. Phe1 was found to have an edge−face geometry relative to ReAsH. The close interaction between Phe1 and ReAsH may be highly significant for the fluorescence properties of the ReAsH complex.

National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-27483 (URN)10.1021/ja809315x (DOI)000264806300030 ()
Available from: 2009-05-05 Created: 2009-05-05 Last updated: 2017-12-13Bibliographically approved
2. Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
Open this publication in new window or tab >>Elucidating cell-penetrating peptide mechanisms of action for membrane interaction, cellular uptake, and translocation utilizing the hydrophobic counter-anion pyrenebutyrate
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2009 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1788, no 12, 2509-2517 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) are membrane permeable vectors recognized for their intrinsic ability to gain access to the cell interior. The hydrophobic counter-anion, pyrenebutyrate, enhances cellular uptake of oligoarginine CPPs. To elucidate CPP uptake mechanisms, the effect of pyrenebutyrate on well-recognized CPPs with various hydrophobicity and arginine content is investigated. The cellular CPP-uptake and CPP-mediated oligonucleotide delivery is analyzed by fluorescence activated cell sorting, confocal microscopy, and a cell based splice-switching assay. The splice-switching oligonucleotide is a mixmer of 2’-O-methyl RNA and locked nucleic acids delivered as a non-covalent complex with 10-fold molar CPP excess. CPP-induced membrane perturbation on large unilamellar vesicles is investigated in calcein release experiments. We observed that pyrenebutyrate facilitates cellular uptake and translocation of oligonucleotide mediated by oligoarginine nonamer while limited effect of pyrenebutyrate on more hydrophobic CPPs was observed. By combining the different experimental results we conclude that the pathway for cellular uptake of oligoarginine is dominated by direct membrane translocation, whereas the pathway for oligoarginine-mediated oligonucleotide translocation is dominated by endocytosis. Both mechanisms are promoted by pyrenebutyrate and we suggest that pyrenebutyrate has different sites of action for the two uptake and translocation mechanisms.

Keyword
cell-penetrating peptide, oligonucleotide delivery, pyrenebutyrate, cellular translocation, locked nucleic acid, splice switching
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology; Biophysics
Identifiers
urn:nbn:se:su:diva-31144 (URN)10.1016/j.bbamem.2009.09.014 (DOI)000272581500005 ()
Projects
Multidisciplinary BIOVINNOVA-SAMBIO
Available from: 2009-11-06 Created: 2009-11-05 Last updated: 2017-12-12Bibliographically approved
3. Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport Across Phospholipid Membranes Induced by a Proton Gradient
Open this publication in new window or tab >>Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport Across Phospholipid Membranes Induced by a Proton Gradient
2011 (English)In: Journal of drug delivery, ISSN 2090-3022, Vol. 2011, 897592- p.Article in journal (Refereed) Published
Abstract [en]

Detergent-mediated reconstitution of bacteriorhodopsin (BR) into large unilamellar vesicles (LUVs) was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG) as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-66760 (URN)10.1155/2011/897592 (DOI)
Available from: 2011-12-21 Created: 2011-12-21 Last updated: 2011-12-28Bibliographically approved
4. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient
Open this publication in new window or tab >>Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient
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2013 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1828, no 4, 1198-1204 p.Article in journal (Refereed) Published
Abstract [en]

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

Keyword
Cell-penetrating peptide, Endosomal escape, Bacteriorhodopsin, Large unilamellar vesicle, Fluorescein-label, Membrane translocation
National Category
Biophysics Chemical Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-84928 (URN)10.1016/j.bbamem.2012.12.008 (DOI)000316522100003 ()23261392 (PubMedID)
Funder
Swedish Research CouncilVINNOVASwedish Foundation for Strategic Research
Available from: 2013-01-03 Created: 2013-01-03 Last updated: 2017-12-06Bibliographically approved
5. Perturbations of model membranes induced by pathogenic dynorphin A mutants causing neurodegeneration in human brain
Open this publication in new window or tab >>Perturbations of model membranes induced by pathogenic dynorphin A mutants causing neurodegeneration in human brain
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 411, no 1, 111-114 p.Article in journal (Refereed) Published
Abstract [en]

Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.

Keyword
Prodynorphin, Dynorphin A, Spinocerebellar ataxia, Non-opioid activity, Large unilamellar vesicle, Calcein leakage
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-66588 (URN)10.1016/j.bbrc.2011.06.105 (DOI)000293368000019 ()
Note
authorCount :6Available from: 2011-12-21 Created: 2011-12-20 Last updated: 2017-12-08Bibliographically approved

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