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Membrane-mimetic systems: Novel methods and results from studies of respiratory enzymes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Peter Brzezinski)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The processes localized to biological membranes are of great interest, both from a scientific and pharmaceutical point of view. Understanding aspects such as the detailed mechanism and regulation of these processes requires investigation of the structure and function of the membrane-bound proteins in which they take place. The study of these processes is often complicated by the need to create in vitro systems that mimic the environment in which these proteins are normally found in vivo. This thesis describes some of the methods available for membrane-protein studies in membrane-mimetic systems, as well as our work aimed at developing such systems. Furthermore, results from studies using these systems are described.

In the first two studies, described in Papers I & II, we investigated the use of silica particle-supported lipid bilayers, both for membrane-protein studies and as possible drug-delivery vehicles. Successful reconstitution of a multisubunit proton-pump, cytochrome c oxidase is described and characterized. Initial attempts to develop drug-delivery systems with two different targeting peptides are also described in the thesis.

The second part of this thesis revolves around our work with membraneprotein dependent pathways. Results from studies of systems where the proton- pump bo3 oxidase and ATP synthase work in concert are described. The results show a surprising lipid-composition dependence for the coupled bo3- ATP-synthase activity (Paper III).

Finally, a new system utilizing synaptic vesicle-fusion proteins for coreconstitution of membrane proteins is described, showing successful coreconstitution of a small respiratory chain, delivery of soluble proteins to preformed liposomes and reconstitution of ATP synthase in native membranes (Paper IV).

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2013. , 55 p.
Keyword [en]
Lipids, membrane proteins, method development, respiration, reconstitution, supported membranes, SNAREs, liposome fusion, lipid-protein interactions
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-94554ISBN: 978-91-7447-774-0 (print)OAI: oai:DiVA.org:su-94554DiVA: diva2:653663
Public defence
2013-11-08, 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 4: Manuscript.

Available from: 2013-10-17 Created: 2013-10-04 Last updated: 2013-10-10Bibliographically approved
List of papers
1. Formation of supported lipid bilayers on silica particles studied using flow cytometry
Open this publication in new window or tab >>Formation of supported lipid bilayers on silica particles studied using flow cytometry
2009 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 25, no 8, 4601-4606 p.Article in journal (Refereed) Published
Abstract [en]

Silica colloidal particles with functionalized surfaces are used, for example, in studies of membrane proteins or for drug delivery, where novel applications are based on the use of particles covered by lipid membrane bilayers. The mechanism by which such supported lipid bilayers are formed on spherical support is not fully understood. Here, we present results from studies of this process using a new method based on flow cytometry. The approach enabled us to detect particle populations coated and uncoated with lipids in the same sample according to the vesicle:particle surface area ratio. The data suggest that DOPC lipid vesicles efficiently break upon interaction with the silica colloidal particle surface; only a small fraction of the adsorbed vesicles remain unbroken. Furthermore, the data support earlier observations showing that formation of the lipid bilayer at the surface is a cooperative process, where bilayer formation is catalyzed by previously bound membrane fragments.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-34729 (URN)10.1021/la8036296 (DOI)000265281700059 ()19265407 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
2. A Membrane-reconstituted Multisubunit Functional Proton Pump on Mesoporous Silica Particles
Open this publication in new window or tab >>A Membrane-reconstituted Multisubunit Functional Proton Pump on Mesoporous Silica Particles
2009 (English)In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 3, no 9, 2639-2646 p.Article in journal (Refereed) Published
Abstract [en]

We have investigated formation of a proteolipid membrane surrounding mesoporous silica particles with a diameter of 550 nm and pore sizes of 3.0 nm. A multisubunit redox-driven proton pump, cytochrome c oxidase, was incorporated into the membrane and we show that the enzyme is fully functional, both with respect to catalysis of O2 reduction to water, and charge separation across the membrane. The orientation of cytochrome c oxidase in the membrane was found to be the same (~70/30 %) in the lipid vesicles and in the silica-particle supported lipid membrane, which provides information on the mechanism by which the vesicles adsorb to the surface. Furthermore, cytochrome c oxidase could maintain a proton electrochemical gradient across the supported proteomembrane, i.e. the membrane system was proton tight, defining an interior particle compartment that is separated from the surrounding aqueous media. Such a biofunctional cellular interface, supported onto a colloid that has a connected interior cytoskeleton-like pore structure, provides a basis for functional studies of membrane-bound transport proteins, and also for applications within pharmaceutical drug delivery.

Keyword
Supported lipid bilayer, mesoporous spheres, nanoparticles, membrane protein, drug delivery, cytochrome c oxidase
National Category
Inorganic Chemistry Industrial Biotechnology Physical Chemistry
Research subject
Biochemistry; Materials Science
Identifiers
urn:nbn:se:su:diva-27829 (URN)10.1021/nn9005413 (DOI)000269988600027 ()
Projects
Synthesis, functionalisation and controlled release of mesoporous materials
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2017-12-13Bibliographically approved
3. Effect of lipid composition on respiration-driven ATP synthesis
Open this publication in new window or tab >>Effect of lipid composition on respiration-driven ATP synthesis
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane. The free energy stored in this gradient is used, for example, for transport of molecules or ions by other transporters as well as for generation of ATP by the ATP synthase. Understanding the overall process requires functional studies of the coupled reactions, which involve co-reconstitution of e.g. a proton pump and a transporter that utilizes the proton gradient. This process is likely to be further influenced by the composition of the membrane, which, for example, may facilitate lateral proton transfer. In the present study, we have co-reconstituted the proton pump bo3 ubiquinol oxidase with ATP synthase, both from E. coli, in liposomes with different membrane compositions. The coupled proton pumping and ATP-synthesis activities were investigated. We found that the ATP synthesis was significantly higher in a membrane composed of pure DOPC lipids than in the presence of DOPA, DOPE, DOPG or cardiolipin. The drop in activity was considerably more pronounced upon addition of the negatively charged head groups (PA, PG or cardiolipin) than upon addition of the zwitterionic PE. The origin of these effects is discussed.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-94552 (URN)
Funder
Swedish Research Council
Available from: 2013-10-04 Created: 2013-10-04 Last updated: 2013-10-07Bibliographically approved
4. SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes
Open this publication in new window or tab >>SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-94553 (URN)
Funder
Swedish Research Council
Available from: 2013-10-04 Created: 2013-10-04 Last updated: 2013-10-07Bibliographically approved

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