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Analysing the morphology of DHPC/DMPC complexes by diffusion NMR
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0002-2713-7731
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Mixtures of lipids and detergents are known to form bicelles at certain parameter ranges, but many un-certainties remain concerning the details of the phase be-haviour of these mixtures and the morphology of the formed lipid assemblies. Here we used NMR diffusion data in com-bination with the multivariate processing method SCORE to analyze mixtures of DHPC and DMPC with the relative concentration q=[DMPC]/[DHPC]=0.5 at total lipid con-centrations from 15 to 300 mM. With this approach we were able to resolve the heavily overlapping mixture spectra into component spectra and obtained reliable diffusion coeffi-cients for lipid concentrations in the range 15 to 200 mM. Between 200 and 300 mM, the similar diffusion coefficients in combination with substantial signal overlap makes it difficult to get very reliable spectra and diffusion coeffi-cients with standard processing parameters, but overfactoring provided useful diffusion coefficient estimates also at these concentrations. At 50–300 mM total lipid concentration, the radii estimated from the diffusion coeffi-cient of DMPC indicate assemblies of the appropriate bicelle size, although small size variations exist, while at lower concentrations the morphology appears to change to larger assemblies. Taken together, the results suggest that for q=0.5 DMPC/DHPC mixtures there is a relatively broad concentration range above 50 mM where bicelles may relia-bly be assumed to adopt the 'classical' bicelle morphology. At lower concentrations there is evidence for a more com-plex morphology with more than one type of lipid assembly in the sample.

National Category
Biophysics
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-107826OAI: oai:DiVA.org:su-107826DiVA: diva2:750691
Available from: 2014-09-29 Created: 2014-09-29 Last updated: 2014-09-30
In thesis
1. The opioid peptide dynorphin A: Biophysical studies of peptide–receptor and peptide–membrane interactions
Open this publication in new window or tab >>The opioid peptide dynorphin A: Biophysical studies of peptide–receptor and peptide–membrane interactions
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA.

The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event.

With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge.

Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 72 p.
Keyword
dynorphin A, opioid receptor, neuropeptide, bicelles, NMR, diffusion, decomposition
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-107766 (URN)978-91-7649-011-2 (ISBN)
Public defence
2014-10-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

Available from: 2014-10-09 Created: 2014-09-29 Last updated: 2014-10-13Bibliographically approved

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