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Protein complexes of the Escherichia coli cell envelope
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Daniel Daley)
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: urn:nbn:se:su:diva-42248ISBN: 978-91-7447-118-2 (print)OAI: oai:DiVA.org:su-42248DiVA: diva2:344563
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
List of papers
1. Protein complexes of the Escherichia coli cell envelope
Open this publication in new window or tab >>Protein complexes of the Escherichia coli cell envelope
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2005 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 41, 34409-34419 p.Article in journal (Refereed) Published
Abstract [en]

Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.

Keyword
Blotting; Western, Cell Cycle Proteins/chemistry/*pharmacology, Cell Division, Cell Membrane/*metabolism, Cytochromes/metabolism, Dimerization, Electron Transport Chain Complex Proteins/metabolism, Electrophoresis; Gel; Two-Dimensional, Electrophoresis; Polyacrylamide Gel, Escherichia coli/metabolism/*physiology, Escherichia coli Proteins/chemistry/metabolism/pharmacology/*physiology, Ions/chemistry, Macromolecular Substances/chemistry, Mass Spectrometry, Membrane Proteins/chemistry, Mutation, Oxidoreductases/chemistry/metabolism/*pharmacology, Protein Binding, Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization
National Category
Biochemistry and Molecular Biology
Research subject
Biogeochemistry
Identifiers
urn:nbn:se:su:diva-18892 (URN)10.1074/jbc.M506479200 (DOI)16079137 (PubMedID)
Available from: 2007-12-27 Created: 2007-12-27 Last updated: 2017-12-13Bibliographically approved
2. Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
Open this publication in new window or tab >>Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
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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.

Keyword
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:nbn:se:su:diva-56162 (URN)10.1021/pr101105c (DOI)000288924000038 ()21210718 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
3. Assembly of the cytochrome bo3 complex
Open this publication in new window or tab >>Assembly of the cytochrome bo3 complex
2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 371, no 3, 765-773 p.Article in journal (Refereed) Published
Abstract [en]

An understanding of the mechanisms that govern the assembly of macromolecular protein complexes is fundamental for studying their function and regulation. With this in mind, we have determined the assembly pathway for the membrane-embedded cytochrome bo3 of Escherichia coli. We show that there is a preferred order of assembly, where subunits III and IV assemble first, followed by subunit I and finally subunit II. We also show that cofactor insertion catalyses assembly. These findings provide novel insights into the biogenesis of this model membrane protein complex.

Keyword
cytochrome bo3, macromolecular assembly, membrane protein, BN-PAGE, heme assembly
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-18643 (URN)10.1016/j.jmb.2007.05.045 (DOI)000248719300016 ()17583738 (PubMedID)
Available from: 2007-12-27 Created: 2007-12-27 Last updated: 2017-12-13Bibliographically approved
4. Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli
Open this publication in new window or tab >>Penicillin-binding protein 5 can form a homo-oligomeric complex in the inner membrane of Escherichia coli
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2011 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 9, 1520-1529 p.Article in journal (Refereed) Published
Abstract [en]

Penicillin-binding protein 5 (PBP5) is a DD-carboxypeptidase, which cleaves the terminal D-alanine from the muramyl pentapeptide in the peptidoglycan layer of Escherichia coli and other bacteria. In doing so, it varies the substrates for transpeptidation and plays a key role in maintaining cell shape. In this study, we have analyzed the oligomeric state of PBP5 in detergent and in its native environment, the inner membrane. Both approaches indicate that PBP5 exists as a homo-oligomeric complex, most likely as a homo-dimer. As the crystal structure of the soluble domain of PBP5 (i.e., lacking the membrane anchor) shows a monomer, we used our experimental data to generate a model of the homo-dimer. This model extends our understanding of PBP5 function as it suggests how PBP5 can interact with the peptidoglycan layer. It suggests that the stem domains interact and the catalytic domains have freedom to move from the position observed in the crystal structure. This would allow the catalytic domain to have access to pentapeptides at different distances from the membrane.

Keyword
membrane protein, homo-oligomer, penicillin-binding protein, peptidoglycan biogenesis
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-62902 (URN)10.1002/pro.677 (DOI)000294383900006 ()
Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-12-08Bibliographically approved
5. The CpxA sensor and other cell envelope proteins respond to bilayer stress in Escherichia coli
Open this publication in new window or tab >>The CpxA sensor and other cell envelope proteins respond to bilayer stress in Escherichia coli
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:su:diva-42285 (URN)
Available from: 2010-08-20 Created: 2010-08-20 Last updated: 2010-08-20Bibliographically approved

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