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New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles
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.
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 4, 1044-1050 p.Article in journal (Refereed) Published
Abstract [en]

Galactolipids are the main structural component of plant chloroplastic (thylakoid) membranes and of blue-green algae cell membranes. The predominant lipids in this class are monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG). We here present a method for the preparation of bicelles that contain these galactolipids together with a characterization of the bicelles, and the lipids within the bicelles. NMR diffusion data show that up to 3096 of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in a q = 0.5 DMPC/DHPC lipid matrix can be replaced with either monogalactosyl-diacylglycerol or digalactosyl-diacylglycerol and that these lipids incorporate into the bicelles. No evidence for phase separation is observed. Bicelles made with monogalactosyl-diacylglycerol are significantly larger than bicelles containing only DMPC, already with only 1096 of the DMPC replaced with the galactolipid. The effect of digalactosyl-diacylglycerol on bicelle size is much smaller. These observations are likely to be correlated with the different bilayer-forming properties of the lipids. Monogalactosyl-diacylglycerol is a non-bilayer-forming lipid, while digalactosyl-diacylglycerol is a bilayer-forming lipid. Both galactolipids display extensive local motion within the bilayer, as evidenced by natural abundance carbon-13 relaxation of the lipid molecules. The sugar headgroup regions are motionally restricted and cannot be described by a model that does not take into account anisotropic reorientation of the sugar units. No significant effect of the galactolipids on DMPC dynamics was observed. Our results indicate that these bicelles may become useful as model membrane mimetic media for studies of galactolipid-protein interactions.

Place, publisher, year, edition, pages
2013. Vol. 117, no 4, 1044-1050 p.
National Category
Physical Chemistry
Research subject
Biophysics
Identifiers
URN: urn:nbn:se:su:diva-88249DOI: 10.1021/jp311093pISI: 000314492300009OAI: oai:DiVA.org:su-88249DiVA: diva2:611165
Funder
Swedish Research Council, 621-2011-5964
Note

AuthorCount:3;

Available from: 2013-03-14 Created: 2013-03-12 Last updated: 2017-09-15Bibliographically 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
2. Taming the Griffin: Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles
Open this publication in new window or tab >>Taming the Griffin: Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biological membranes form a protective barrier around cells and cellular compartments. A broad range of biochemical processes occur in or at membranes demonstrating that they are not only of structural but also of functional importance. One important class of membrane proteins are membrane-associated glycosyltransferases. WaaG is a representative of this class of proteins; its function is to catalyze one step in the synthesis of lipopolysaccharides, which are outer membrane lipids found in Gram-negative bacteria.

To study protein-membrane complexes by biophysical methods, one must employ membrane mimetics, i.e. simplifications of natural membranes. One type of membrane mimetic often employed in solution-state NMR is small isotropic bicelles, obloid aggregates formed from a lipid bilayer that is dissolved in aqueous solvent by detergent molecules that make up the rim of the bicelle.

In this thesis, fast dynamics of lipid atoms in bicelles containing lipid mixtures that faithfully mimic plant and bacterial membranes were investigated by NMR relaxation. Lipids were observed to undergo a broad range of motions; while the glycerol backbone was found to be rigid, dynamics in the acyl chains were much more rapid and unrestricted. Furthermore, by employing paramagnetic relaxation enhancements an ‘atomic ruler’ was developed that allows for measurement of the immersion depths of lipid carbon atoms.

WaaG is a membrane-associated protein that adopts a GT-B fold. For proteins of this type, it has been speculated that the N-terminal domain anchors tightly to the membrane via electrostatic interactions, while the anchoring of the C-terminal domain is weaker. Here, this model was tested for WaaG. It was found by a set of circular dichroism, fluorescence, and NMR techniques that an anchoring segment located in the N-terminal domain termed MIR-WaaG binds electrostatically to membranes, and the structure and localization of isolated MIR-WaaG inside micelles was determined. Full-length WaaG was also found to bind membranes electrostatically. It senses the surface charge density of the membrane whilst not discriminating between anionic lipid species. Motion of the C-terminal domain could not be observed under the experimental conditions used here. Lastly, the affinity of WaaG to membranes is lower than expected, indicating that WaaG should not be classified as a monotopic membrane protein but rather as a peripheral one.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017. 77 p.
Keyword
membrane, bicelle, lipid, detergent, lipopolysaccharide, glycosyltransferase, WaaG, fluorescence, circular dichroism, NMR, paramagnetic relaxation enhancement, model-free approach, dynamics
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-146872 (URN)978-91-7649-978-8 (ISBN)978-91-7649-979-5 (ISBN)
Public defence
2017-11-03, 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 5: Manuscript.

Available from: 2017-10-11 Created: 2017-09-14 Last updated: 2017-10-05Bibliographically approved

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