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Hairpin Structure of a Biarsenical−Tetracysteine Motif Determined by NMR Spectroscopy
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2009. Vol. 131, no 13, 4613-4615 p.
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-27483DOI: 10.1021/ja809315xISI: 000264806300030OAI: oai:DiVA.org:su-27483DiVA: diva2:214459
Available from: 2009-05-05 Created: 2009-05-05 Last updated: 2011-12-28Bibliographically approved
In thesis
1. Biophysical studies of membrane associated peptides
Open this publication in new window or tab >>Biophysical studies of membrane associated peptides
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A large part of the processes in living organisms involves proteins acting in a biological membrane. Biophysical studies on isolated model systems can give important understandings of the complicated biological mechanisms in the membrane. In this thesis peptide membrane interaction mechanisms are studied in several different systems. The membrane interactions of the unstructured endogenous opioid peptides dynorphinA (DynA) and dynorphinB (DynB) were investigated with Saturation Transfer Difference (STD) experiments, supplemented by various other NMR methods. The combined results support a conclusion that DynA binds to the lipid bilayer with the N-terminal residues inserted into the hydrophobic region and the C-terminal residues more loosely attached to the surface, while DynB is situated parallel to the bilayer. This difference in membrane interaction can explain observations that DynA has membrane perturbing effects while DynB has not. In the second study the binding domain of the glycosyltransferase A.laidlawii Monoglycosyldiacyl Glycerol Synthase (alMGS) was predicted and investigated mainly with NMR which enabled the determination of the 3D structure and position in a lipid environment. The phospholipid bilayers induce a large amphipathic α-helical content in the peptide, which aligns parallel but slightly tilted along the lipid surface with the N-terminus situated closer to the hydrophobic region. Lipid perturbation effects caused by peptide-membrane interactions were investigated by studying the influence of model transmembrane peptides on lipid dynamics in phospholipid bicelles with varying bilayer thickness. 13C-relaxation NMR of the lipids was used to survey the effects of the model peptides on the lipid bilayer.In paper IV and V structure and membrane interaction properties of the highly charged and flexible helix-turn-helix motif named the 'voltage sensor paddle' from two transmebrane voltage gated potassium channels was investigated. In membrane mimetic media, the KvaP paddle adopts the same type of helix-turn-helix conformation which can be seen in the Xray structure of the entire ion channel. The membrane interaction of the paddle HsapBK was compared with the corresponding one in KvaP, and both were inserted in the lipid bilayer but perturbed the lipid system differently, which may indicate differences in their function. Paper VI treats the structure of the novel site-specific fluorophore ReAsH bound to an optimized peptide sequence. The analysis shows that the important peptide mid segment configuration of CCPGCC is optimal for the ReAsH binding and that the N-terminal Phe1 plays an important role for the fluorophore process.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 55 p.
Keyword
NMR, spectroscopy, membrane interaction, bicelles
National Category
Chemical Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-27488 (URN)978-91-7155-888-6 (ISBN)
Public defence
2009-06-04, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2009-05-14 Created: 2009-05-05 Last updated: 2012-08-28Bibliographically approved
2. Biophysical studies of peptides with functions in biotechnology and biology
Open this publication in new window or tab >>Biophysical studies of peptides with functions in biotechnology and biology
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
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:nbn:se:su:diva-66948 (URN)978-91-7447-417-6 (ISBN)
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

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