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Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
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.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
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2012 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 21, no 10, 1571-1576 p.Article in journal (Refereed) Published
Abstract [en]

A topology map of a membrane protein defines the location of transmembrane helices and the orientation of soluble domains relative to the membrane. In the absence of a high-resolution structure, a topology map is an essential guide for studying structurefunction relationships. Although these maps can be predicted directly from amino acid sequence, the predictions are more accurate if combined with experimental data, which are usually obtained by fusing a reporter protein to the C-terminus of the protein. However, as reporter proteins are large, they cannot be used to report on the cytoplasmic/periplasmic location of the N-terminus of a protein. Here, we show that the bimolecular split-green fluorescent protein complementation system can overcome this limitation and can be used to determine the location of both the N- and C-termini of inner membrane proteins in Escherichia coli.

Place, publisher, year, edition, pages
2012. Vol. 21, no 10, 1571-1576 p.
Keyword [en]
topology, membrane protein, split-GFP, inner membrane
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-82120DOI: 10.1002/pro.2131ISI: 000308994300015OAI: oai:DiVA.org:su-82120DiVA: diva2:566757
Note

AuthorCount:6;

Available from: 2012-11-09 Created: 2012-11-08 Last updated: 2017-12-07Bibliographically approved
In thesis
1. The social life of a membrane protein; It's complex
Open this publication in new window or tab >>The social life of a membrane protein; It's complex
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are key players in many biological processes. Since most membrane proteins are assembled into oligomeric complexes it is important to understand how they interact with each other. Unfortunately however, the assembly process (i.e. their social life) remains poorly understood. In the work presented in this thesis I have investigated when and how membrane proteins assemble with each other and their cofactors to form functional units. We have shown that that cofactor insertion in the hetero-tetrameric cytochrome bo3 occurs at an early state in the assembly process. We also found that the pentameric CorA magnesium ion channel is stabilised by different interactions depending on the magnesium ion concentration in the cell. These studies indicate that the assembly of a functional unit is a dynamic process, which is a result of many different forces. By studying the assembly of membrane proteins we have obtained a deeper insight into their function, which cannot be explained by static crystal structures.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 52 p.
Keyword
membrane proteins, assembly, cofactors, magnesium, Escherichia coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-88597 (URN)978-91-7447-671-2 (ISBN)
Public defence
2013-05-03, 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 2: Manuscript.

Available from: 2013-04-11 Created: 2013-03-21 Last updated: 2013-03-29Bibliographically approved
2. Fluorescence Studies of Cell Division in Escherichia coli
Open this publication in new window or tab >>Fluorescence Studies of Cell Division in Escherichia coli
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Escherichia coli the cell division is carried out by a large dynamic protein complex called the divisome. The divisome assembles in a two-step manner starting with the localization of the eukaryotic tubulin homologue FtsZ to the midcell. Together with other early arriving proteins FtsZ form an intermediate structure called the Z-ring. After a considerable time lag the divisome maturates fully by recruiting several other late arriving proteins before it starts to constrict the cell envelope that ultimately will lead to cytokinesis and the formation of two identical daughter cells. Despite of being objectives of extensive study over the last decades, understanding of the exact molecular roles of many of the divisome proteins is still lacking and to date there is very limited knowledge of the disassembly process of the divisome. In this thesis I have used various fluorescence microscopy based methods to better characterize the role of FtsZ and other divisome proteins during the final stages of the cell division. I have shown that FtsZ disassembles from the divisome prior to inner membrane closure indicating that it is not the force generator during this final step of division that it is widely thought to be. I have also shown that the disassembly of the divisome is a multistep process in which the proteins that arrive in the second step of divisome assembly also remain at the division septum longer than those proteins that arrive in the first step. These findings add new important information regarding the cell division and together they provide a more complete picture of this event that ultimately may lead to more efficient identification of novel antibiotic targets.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 84 p.
Keyword
E. coli, Cell division, FtsZ, Fluorescence microscopy, FRAP
National Category
Biophysics
Research subject
Biophysics
Identifiers
urn:nbn:se:su:diva-102619 (URN)978-91-7447-852-5 (ISBN)
Public defence
2014-06-03, 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 3: Manuscript.

Available from: 2014-05-12 Created: 2014-04-11 Last updated: 2014-05-13Bibliographically approved
3. Engineering membrane proteins for production and topology
Open this publication in new window or tab >>Engineering membrane proteins for production and topology
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli.

For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 70 p.
Keyword
Membrane protein, Over-expression, Protein production, Codon optimization, Escherichia coli, AraH, NarK, mRNA secondary structure, coding sequence, ribosome binding site, RBS, topology, split-GFP
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-116598 (URN)978-91-7649-121-8 (ISBN)
Public defence
2015-05-28, Nordenskiöldsalen, Geovetenskapens Hus, Svante Arrhenius väg 12, Stockholm, 09:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2015-05-06 Created: 2015-04-22 Last updated: 2015-06-24Bibliographically approved

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