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Taming the Griffin: Membrane interactions of peripheral and monotopic glycosyltransferases and dynamics of bacterial and plant lipids in bicelles
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-4057-6699
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. , p. 77
Keyword [en]
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: urn:nbn:se:su:diva-146872ISBN: 978-91-7649-978-8 (print)ISBN: 978-91-7649-979-5 (electronic)OAI: oai:DiVA.org:su-146872DiVA, id: diva2:1141234
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
List of papers
1. New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles
Open this publication in new window or tab >>New Membrane Mimetics with Galactolipids: Lipid Properties in Fast-Tumbling Bicelles
2013 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 117, no 4, p. 1044-1050Article 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.

National Category
Physical Chemistry
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-88249 (URN)10.1021/jp311093p (DOI)000314492300009 ()
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
2. Fast-tumbling bicelles constructed from native Escherichia coli lipids
Open this publication in new window or tab >>Fast-tumbling bicelles constructed from native Escherichia coli lipids
Show others...
2016 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1858, no 9, p. 2097-2105Article in journal (Refereed) Published
Abstract [en]

Solution-state NMR requires small membrane mimetic systems to allow for acquiring high-resolution data. At the same time these mimetics should faithfully mimic biological membranes. Here we characterized two novel fast-tumbling bicelle systems with lipids from two Escherichia coli strains. While strain 1 (AD93WT) contains a characteristic E. coli lipid composition, strain 2 (AD93-PE) is not capable of synthesizing the most abundant lipid in E. coli, phosphatidylethanolamine. The lipid and acyl chain compositions were characterized by P-31 and C-13 NMR. Depending on growth temperature and phase, the lipid composition varies substantially, which means that the bicelle composition can be tuned by using lipids from cells grown at different temperatures and growth phases. The hydrodynamic radii of the bicelles were determined from translational diffusion coefficients and NMR spin relaxation was measured to investigate lipid properties in the bicelles. We find that the lipid dynamics are unaffected by variations in lipid composition, suggesting that the bilayer is in a fluid phase under all conditions investigated here. Backbone glycerol carbons are the most rigid positions in all lipids, while head-group carbons and the first carbons of the acyl chain are somewhat more flexible. The flexibility increases down the acyl chain to almost unrestricted motion at its end. Carbons in double bonds and cyclopropane moieties are substantially restricted in their motional freedom. The bicelle systems characterized here are thus found to faithfully mimic E. coli inner membranes and are therefore useful for membrane interaction studies of proteins with E. coli inner membranes by solution-state NMR.

Keyword
Native lipids, Bicelle, Model-free approach, Dynamics, Diffusion, Inner membrane, Lipid composition
National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-133365 (URN)10.1016/j.bbamem.2016.06.008 (DOI)000380601500016 ()27317394 (PubMedID)
Available from: 2016-09-13 Created: 2016-09-06 Last updated: 2017-09-14Bibliographically approved
3. Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement
Open this publication in new window or tab >>Immersion Depths of Lipid Carbons in Bicelles Measured by Paramagnetic Relaxation Enhancement
2017 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 121, no 32, p. 7660-7670Article in journal (Refereed) Published
Abstract [en]

Myriads of biological processes occur in or at cellular lipid membranes. Knowledge about the localization of proteins, lipids, and other molecules within biological membranes is thus crucial for the understanding of such processes. Here, we present a method to determine the immersion depths of lipid carbon atoms in membranes by paramagnetic relaxation enhancement (PRE) caused by the presence of doxylated lipids. As membrane mimetics, we employ small isotropic bicelles made of synthetic lipids and of natural Escherichia coli phospholipid extract. Bicelles are particularly suitable for solution state NMR since they maintain a lipid bilayer while they are at the same time amenable to solution state NMR experiments. PREs were measured in the presence of different doxylated lipids with the nitroxide radical located in the headgroup and at various positions in the acyl chain. Theoretical PREs were calculated assuming a simple bicelle model using the Solomon–Bloembergen equations. Immersion depths of the lipid carbon atoms were obtained by a least-squares fit of the theoretical to the experimental PREs. The carbon immersion depths correspond well to results obtained by other methods and differences do not exceed 3–5 Å. This means that the method presented here provides sufficient resolution to distinguish the localization of carbons in different regions of the lipid bilayer, despite considerable simplifications of the underlying theory. These simplifications include a simple form of the spectral density function, which we find is sufficient to reliably determine immersion depths. A more complicated spectral density function that includes bicelle, lipid, and local motions may only improve the results if its parametrization is good enough. The approach presented here may be extended to the determination of protein localization in membranes employing realistic membrane mimetics like the bicelles made of E. coli phospholipid extract used here.

National Category
Biochemistry and Molecular Biology Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-146714 (URN)10.1021/acs.jpcb.7b05822 (DOI)000408179800014 ()
Available from: 2017-09-07 Created: 2017-09-07 Last updated: 2017-09-18Bibliographically approved
4. Membrane Interaction of the Glycosyltransferase WaaG
Open this publication in new window or tab >>Membrane Interaction of the Glycosyltransferase WaaG
2015 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 109, no 3, p. 552-563Article in journal (Refereed) Published
Abstract [en]

The glycosyltransferase WaaG is involved in the synthesis of lipopolysaccharides that constitute the outer leaflet of the outer membrane in Gram-negative bacteria such as Escherichia coli. WaaG has been identified as a potential antibiotic target, and inhibitor scaffolds have previously been investigated. WaaG is located at the cytosolic side of the inner membrane, where the enzyme catalyzes the transfer of the first outer-core glucose to the inner core of nascent lipopolysaccharides. Here, we characterized the binding of WaaG to membrane models designed to mimic the inner membrane of E. coli. Based on the crystal structure, we identified an exposed and largely a-helical 30-residue sequence, with a net positive charge and several aromatic amino acids, as a putative membrane-interacting region of WaaG (MIR-WaaG). We studied the peptide corresponding to this sequence, along with its bilayer interactions, using circular dichroism, fluorescence quenching, fluorescence anisotropy, and NMR. In the presence of dodecylphosphocholine, MIR-WaaG was observed to adopt a three-dimensional structure remarkably similar to the segment in the crystal structure. We found that the membrane interaction of WaaG is conferred at least in part by MIR-WaaG and that electrostatic interactions play a key role in binding. Moreover, we propose a mechanism of anchoring WaaG to the inner membrane of E. coli, where the central part of MIR-WaaG inserts into one leaflet of the bilayer. In this model, electrostatic interactions as well as surface-exposed Tyr residues bind WaaG to the membrane.

National Category
Biological Sciences
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-120191 (URN)10.1016/j.bpj.2015.06.036 (DOI)000359180400012 ()
Available from: 2015-09-04 Created: 2015-09-02 Last updated: 2017-12-04Bibliographically approved
5. The glycosyltransferase WaaG: a peripheral membrane protein?
Open this publication in new window or tab >>The glycosyltransferase WaaG: a peripheral membrane protein?
(English)Manuscript (preprint) (Other academic)
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-146870 (URN)
Available from: 2017-09-14 Created: 2017-09-14 Last updated: 2017-09-15Bibliographically approved

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