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Native incorporation of outer membrane proteins into vesicles suitable for high resolution NMR spectroscopy
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
2018 (English)Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
Abstract [en]

Nuclear Magnetic Resonance spectroscopy is a powerful tool for studying the structure and dynamics of membrane proteins. The method has mainly been used for membrane proteins solubilized in detergent or embedded in membrane mimetic systems. Detergents/membrane mimetics do not ensure glycolipid polarity and native lipid distribution, resulting in the questionable biological relevance of data. Therefore, the aim of this project was to develop a method for collection of Nuclear Magnetic Resonance data from membrane proteins in a native lipid architecture.

To achieve this, we use a special Escherichia coli strain that was engineered to shed off its outer membrane at a high rate. The protein of interest is designed with a translocation-tag that takes it to the outer membrane and is concomitantly shed-off during growth and expression. The vesicle-membrane is then precipitated directly from bacterial culture media, resuspended in buffer and concentrated. Nuclear Magnetic Resonance spectroscopy can be directly performed on the sample or it can be stored for later use.

We could show the success of the method using a model protein (OmpW) by measuring Nuclear Magnetic Resonance spectra from OmpW containing vesicles as well as from OmpW in detergent micelles. This method has the potential to significantly shape future structural and dynamic studies of membrane proteins and is likely going to have a huge impact on our understanding of host-pathogen interactions and drug development at a molecular level.

Place, publisher, year, edition, pages
2018. , p. 45
National Category
Biophysics
Identifiers
URN: urn:nbn:se:uu:diva-355455OAI: oai:DiVA.org:uu-355455DiVA, id: diva2:1229130
Educational program
Master Programme in Molecular Biotechnology
Supervisors
Examiners
Available from: 2018-06-29 Created: 2018-06-29 Last updated: 2018-06-29Bibliographically approved

Open Access in DiVA

The full text will be freely available from 2020-06-05 11:58
Available from 2020-06-05 11:58

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