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Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
Stockholm University, Faculty of Science, Department of Analytical Chemistry.
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.
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2011 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 10, no 4, 1848-1859 p.Article in journal (Refereed) Published
Abstract [en]

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.

Place, publisher, year, edition, pages
2011. Vol. 10, no 4, 1848-1859 p.
Keyword [en]
Escherichia coli, cell envelope, proteome, membrane protein, protein complex, BN-PAGE, PpiD, YfgM
National Category
Analytical Chemistry Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-56162DOI: 10.1021/pr101105cISI: 000288924000038PubMedID: 21210718OAI: oai:DiVA.org:su-56162DiVA: diva2:409894
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Strategies to explore the membrane proteome of a cell
Open this publication in new window or tab >>Strategies to explore the membrane proteome of a cell
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope plays key roles in numerous processes such as maintaining cellular integrity, communication with other cells, signal transduction, maintenance of cellular homeostasis, and regulation of the traffic of molecules between the cell and the extracellular milieu. Essential membrane components in many of these processes are proteins. It is estimated that ~20-30% of the predicted open reading frames (ORFs) of all organisms encode membrane proteins. Furthermore, two thirds of drug targets are membrane proteins. However, despite their importance, membrane proteins have so far been mostly neglected in most proteomic studies, due to the inherent challenges in analyzing them.

The focus of this thesis is to devise strategies that allow investigation of membrane proteins and their associated complexes. Optimization of sample preparation in the underlying studies has allowed important goals to be reached in membrane protein analyses at various levels such as elucidation of their primary structure by collision-induced dissociation (CID) and electron-capture dissociation (ECD) mass spectrometry (MS), profiling membrane proteins and their complexes, the discovery of novel protein complexes, definition of their topology, and unambiguous identification of protein-bound ligand(s). This thesis paves the way for better characterization of membrane proteins and their assemblies hinting towards the crucial role(s) they play in maintaining normal cell physiology.

Place, publisher, year, edition, pages
Stockholm: Department of Analytical Chemistry, Stockholm University, 2011. 94 p.
Keyword
Membrane proteins, Proteomics, Escherichia coli, Enterococcus faecalis, Synechocystis, Mass Spectrometry, BN/SDS-PAGE, FT-ICR-MS, Myelin P2 protein
National Category
Analytical Chemistry
Research subject
Analytical Chemistry
Identifiers
urn:nbn:se:su:diva-56848 (URN)978-91-7447-211-0 (ISBN)
Public defence
2011-06-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript. Available from: 2011-05-12 Created: 2011-04-28 Last updated: 2012-01-24Bibliographically approved
2. Protein complexes of the Escherichia coli cell envelope
Open this publication in new window or tab >>Protein complexes of the Escherichia coli cell envelope
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope of Escherichia coli, as for all living cells, is a magnificent semi-permeable membrane barrier that facilitates protection as well as enables fundamental contact with the exterior world. The envelope comprises a mixture of phospholipids, organized in two bilayers, which are stabilized by a rigid peptidoglycan layer. There are also a large number of proteins, which can be lipid-integrated or attached. Infact, it is anticipated that approximately 30-40% of the cellular proteome of E. coli could be associated with the envelope. These proteins are involved in the transport of small molecules and nutrients, the biogenesis of the envelope, metabolism, signaling, channeling and cellular movement and attachment.

The focus of this thesis is to understand the cell envelope of E. coli by understanding the proteins it holds. Three main questions have been addressed: 1) Which proteins are present? 2) How do these proteins interact? 3) How are the interactions brought about? To answer these questions we have designed and optimized methods suitable for proteome-wide separation, visualization and characterization of membrane proteins and protein complexes. We present reference proteome and interactome maps of the envelope, which further our understanding of the assembly and composition of the cell envelope. In many instances our studies have provided a first step towards understanding protein function(s) and for carrying out meaningful biochemical and structural analysis. We have also developed parallel approaches, which have enabled us to dissect the assembly process for two specific membrane protein complexes, a homo-dimer of penicillin binding protein 5 and the respiratory oxidase cytochrome bo3. These studies have extended our understanding of the relationship between structure and function of protein complexes.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2010. 82 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42248 (URN)978-91-7447-118-2 (ISBN)
Public defence
2010-10-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 14:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript. Paper 5: Manuscript.

Available from: 2010-09-20 Created: 2010-08-19 Last updated: 2014-08-01Bibliographically approved
3. Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone
Open this publication in new window or tab >>Protein trafficking in the cell envelope of Escherichia coli: Identification and characterisation of a novel chaperone
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cell envelope of Gram-negative bacteria, like Escherichia coli, is composed of a cytoplasmic membrane, a periplasmic space containing a peptidoglycan layer and an outer membrane. About 30 % of all proteins are localised in the cell envelope. These proteins have to be inserted into or translocated across the inner membrane by the SecYEG translocon. They are then chaperoned to their final destination by a network of chaperones. The broad aim of this work was to provide a better understanding of protein trafficking through the bacterial cell envelope. We have identified a novel membrane protein complex consisting of the periplasmic chaperone PpiD and the uncharacterised protein YfgM. Both are anchored in the inner membrane and have periplasmic domains. By co-immunoprecipitations and two-dimensional gel electrophoresis it could be demonstrated that YfgM and PpiD form a supercomplex with the SecYEG translocon. Furthermore, a chemical-genetic approach showed that YfgM is part of the periplasmic chaperone network that is essential for envelope protein biogenesis. Moreover, it could be shown that YfgM is required for the stability of the periplasmic chaperone HdeB. Finally, evidence that YfgM might also be involved in the lateral insertion of transmembrane domains was provided. In summary, this thesis details the identification and characterisation of a novel ancillary subunit of the SecYEG translocon that is involved in the periplasmic chaperone network in the cell envelope of Escherichia coli.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2014. 73 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-106277 (URN)978-91-7447-944-7 (ISBN)
Public defence
2014-09-25, 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: 2014-09-03 Created: 2014-07-30 Last updated: 2017-10-30Bibliographically approved
4. 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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