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The social life of a membrane protein; It's complex
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
membrane proteins, assembly, cofactors, magnesium, Escherichia coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-88597ISBN: 978-91-7447-671-2 (print)OAI: oai:DiVA.org:su-88597DiVA: diva2:612330
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At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.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
List of papers
1. Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
Open this publication in new window or tab >>Application of split-green fluorescent protein for topology mapping membrane proteins in Escherichia coli
Show others...
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.

Keyword
topology, membrane protein, split-GFP, inner membrane
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-82120 (URN)10.1002/pro.2131 (DOI)000308994300015 ()
Note

AuthorCount:6;

Available from: 2012-11-09 Created: 2012-11-08 Last updated: 2017-12-07Bibliographically approved
2. Why is the GMN-motif in the CorA/Mrs2/Alr1 superfamily of magnesium channels conserved?
Open this publication in new window or tab >>Why is the GMN-motif in the CorA/Mrs2/Alr1 superfamily of magnesium channels conserved?
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-88596 (URN)
Available from: 2013-03-21 Created: 2013-03-21 Last updated: 2013-03-29Bibliographically approved
3. The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel
Open this publication in new window or tab >>The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 33, 27547-27555 p.Article in journal (Refereed) Published
Abstract [en]

Crystal structures of the CorA Mg2+ channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open metal free state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg2+. We find that when Mg2+ is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg2+ is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-82951 (URN)10.1074/jbc.M112.371484 (DOI)000307840700030 ()
Note

AuthorCount:3;

Available from: 2012-12-14 Created: 2012-12-03 Last updated: 2017-12-06Bibliographically approved
4. Heme incorporation into the cytochrome bo(3) occurs at a late stage of assembly
Open this publication in new window or tab >>Heme incorporation into the cytochrome bo(3) occurs at a late stage of assembly
2012 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, no 23, 4197-4202 p.Article in journal (Refereed) Published
Abstract [en]

Respiratory complexes in both prokaryotes and eukaryotes contain multiple co-factors, which are coordinated in defined positions so that they can function as electron wires. Intriguingly, co-factors are usually buried deep within hetero-oligomeric protein complexes and it is not clear when or how they are incorporated. In this study we show that heme is incorporated into the cytochrome bo(3) complex of Escherichia coli at a late stage of assembly. Specifically the apo-form of subunit I (the catalytic subunit) interacts with subunits III and IV before accepting heme. Assembly of subunit II is stalled until heme is incorporated.

Keyword
Respiratory complex, Membrane protein assembly, Co-factor insertion, Heme, BN-PAGE, Escherichia coli inner membrane
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-84797 (URN)10.1016/j.febslet.2012.10.021 (DOI)000311346100022 ()
Note

AuthorCount:2;

Available from: 2013-01-02 Created: 2013-01-02 Last updated: 2017-12-06Bibliographically approved

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