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A Membrane-reconstituted Multisubunit Functional Proton Pump on Mesoporous Silica Particles
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
Stockholm University, Faculty of Science, Department of Physical, Inorganic and Structural Chemistry.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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.

Place, publisher, year, edition, pages
2009. Vol. 3, no 9, 2639-2646 p.
Keyword [en]
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: urn:nbn:se:su:diva-27829DOI: 10.1021/nn9005413ISI: 000269988600027OAI: oai:DiVA.org:su-27829DiVA: diva2:218562
Projects
Synthesis, functionalisation and controlled release of mesoporous materials
Available from: 2009-05-20 Created: 2009-05-20 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Harnessing Mesoporous Spheres - transport studies and biotechnological applications
Open this publication in new window or tab >>Harnessing Mesoporous Spheres - transport studies and biotechnological applications
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Applications in controlled release and delivery calls for a good understanding of molecular transport within the carrier material and the dominating release mechanisms. It is clear that a better understanding of hindered transport and diffusion of guest molecules is important when developing new porous materials, e.g., surfactant templated silica spheres, for biotechnological applications. Confocal laser scanning microscopy was used to quantify the bulk release and intraparticle transport of small charged fluorescent dyes, and fluorescently-tagged neutral dextran, from mesoporous silica spheres. The time dependent release and the concentration profiles within the spheres have been used to analyze the release mechanisms using appropriate models. While the small, non-adsorbing anionic dye is released following a simple diffusion driven process, the concentration of the cationic dye varies radially within the spheres after loading. The release of the cationic dye is controlled by diffusion after an initial period of rapid release, which could be due to a significant fraction of the cationic dye that remains permanently attached to the negatively charged walls of the mesoporous silica spheres. The diffusion of dextran and the resulting flat concentration profiles could be related to the complex structural feature of the cylindrical pores close to the surface, and a possible conformational change of the dextran with the concentration. The stability and leaching of a catalytic enzyme, lipase, immobilized in hydrophobilized mesoporous support has also been quantified. Colloidal monodisperse mesoporous silica spheres were synthesized and transmission electron microscopy showed that the inner pore structure display a radially extending pores. The mesoporous spheres were used as solid supports for a lipid membrane incorporated with a multi-subunit redox-driven proton pump, which was shown to remain functional.

Place, publisher, year, edition, pages
Stockholm: Department of Physical, Inorganic and Structural Chemistry, Stockholm University, 2009. 69 p.
Keyword
Mesoporous, spheres, particles, CLSM, TEM, controlled-release, molecular transport, lipid membrane, enzyme, intraparticle, lipase, solid support
National Category
Inorganic Chemistry
Research subject
Materials Science
Identifiers
urn:nbn:se:su:diva-27797 (URN)978-91-7155-898-5 (ISBN)
Public defence
2009-09-02, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 8 C, Stockholm, 10:00 (English)
Opponent
Supervisors
Projects
Synthesis, functionalisation and controlled release of mesoporous materials
Available from: 2009-05-27 Created: 2009-05-19 Last updated: 2009-05-20Bibliographically approved
2. Membrane-mimetic systems: Novel methods and results from studies of respiratory enzymes
Open this publication in new window or tab >>Membrane-mimetic systems: Novel methods and results from studies of respiratory enzymes
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
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:nbn:se:su:diva-94554 (URN)978-91-7447-774-0 (ISBN)
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

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