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Characterization of the Morphology of Fast-Tumbling Bicelles with Varying Composition
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Karolinska Institutet, Sweden.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 30, no 19, 5488-5496 p.Article in journal (Refereed) Published
Abstract [en]

Small, fast-tumbling bicelles are frequently used in solution NMR studies of protein lipid interactions. For this purpose it is critical to have information about the organization of the lipids within the bicelle structure. We have studied the morphology of small, fast-tumbling bicelles containing DMPC and DHPC as a function of temperature, lipid concentration, and the relative ratio (q value) of lipid (DMPC) to detergent (DHPC) amounts. Dynamic light scattering and cryo-transmission electron microscopy techniques were used to measure the size of the bicelles and to monitor the shape and dispersity of the particles in the samples. The stability and size of DMPC-containing bicelle mixtures were found to be highly dependent on temperature and the total lipid concentration for mixtures with q = 1 and q = 1.5. Stable DMPC/DHPC bicelles are only formed at low q values (0.5). Bicelle mixtures with q > 0.5 appear to be multidisperse containing more than one component, one with r(H) around 2.5 nm and one with r(H) of 6-8 nm. This is interpreted as a coexistence of small (possibly mixed micelles) bicelles and much larger bicelles. Incubating the sample at 37 degrees C increases the phase separation. Moreover, low total amphiphile concentrations and low q values lead to the formation of a temperature-independent morphology, interpreted as the formation of small particles in which the DHPC and DMPC are more mixed. On the basis of these results, we propose the existence of a critical bicelle concentration, a parameter that determines the existence of bilayered bicelles, which varies with q value. This polymorphism was not observed at any concentrations for q = 0.5 bicelles, for which a small but detectable temperature dependence was observed at high concentrations. The results demonstrate that q = 0.5 mixtures predominantly form classical bicelles, but that caution is needed when using fast-tumbling mixtures with q values higher than 0.5.

Place, publisher, year, edition, pages
2014. Vol. 30, no 19, 5488-5496 p.
National Category
Chemical Sciences
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-105194DOI: 10.1021/la500231zISI: 000336414500019OAI: oai:DiVA.org:su-105194DiVA: diva2:732495
Funder
Swedish Research Council
Note

AuthorCount:4;

Available from: 2014-07-04 Created: 2014-06-24 Last updated: 2017-12-05Bibliographically approved
In thesis
1. One key to two doors: Dual targeting peptides and membrane mimetics
Open this publication in new window or tab >>One key to two doors: Dual targeting peptides and membrane mimetics
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A targeting peptide at the N-terminus of a precursor protein usually directs the protein synthesized in the cytosol to a specific organelle in the cell. Interestingly, some targeting peptides, so-called dual targeting peptides (dTPs) can target their protein to both mitochondria and chloroplasts. In order to understand the mechanism of dual targeting, a dTP from threonyl tRNA synthetase (ThrRS-dTP) was investigated as a model dTP in this thesis work. The results suggest that ThrRS-dTP is intrinsically disordered in solution but has an α-helical propensity at the N-terminal part. Tom20 and Toc34 are the two primary receptors on the outer membranes of mitochondria and chloroplasts, respectively. We found that the N-terminal half of the ThrRS-dTP sequence, including an amphiphilic helix, is important for the interaction with Tom20. This part also contains a φχχφφ motif, where φ represents a hydrophobic/aromatic residue and χ represents any amino acid residue. In contrast, neither the amphiphilic helix nor φχχφφ motif in ThrRS-dTP has any special role for its interaction with Toc34. Instead, the entire sequence of ThrRS-dTP is important for Toc34 interaction, including the C-terminal part which is barely affected by Tom20 interaction.

In addition, the role of lipids in the organelle membrane for the recognition of dual targeting peptides during protein import is also the focus of this thesis. The tendency to form α-helix in ThrRS-dTP, which is not observable in solution by CD, becomes obvious in the presence of lipids and DPC micelles. To be able to study such interactions, DMPC/DHPC isotropic bicelles under different conditions have also been characterized. These results demonstrate that bicelles with a long-chained/short-chained lipid ratio q = 0.5 and a concentration larger than 75 mM should be used to ensure that the classic bicelle morphology persists. Moreover, we developed a novel membrane mimetic system containing the galactolipids, MGDG or DGDG, which have been proposed to be important for protein import into chloroplasts. Up to 30% MGDG or DGDG lipids were able to be integrated into bicelles. The local dynamics of the galactolipids in bicelles displays two types of behavior: the sugar head-group and the glycerol part are rigid, and the acyl chains are flexible.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 69 p.
Keyword
: Dual targeting peptides, protein import, mitochondria and chloroplasts, bicelles, galactolipids, NMR spectroscopy
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-116817 (URN)978-91-7649-159-1 (ISBN)
Public defence
2015-05-29, Magnéli hall, Arrhenius Laboratory, 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 2: In press.

Available from: 2015-05-07 Created: 2015-04-28 Last updated: 2015-06-29Bibliographically approved

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