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Membrane protein topogenesis
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The membranes of cells are highly complex and heterogeneous structures that fulfill multiple vital tasks. They form thin barriers that seal out the environment, thus defining the cell’s boundaries. They mediate the selective exchange of information and substances between the inside and outside of cells, thus making cellular life and survival possible and allowing fast adaptation to changing conditions. Not least importantly, they harbor key components of many essential processes such as the photosynthesis and respiration. Membranes are composed of a largely apolar lipid matrix densely punctuated with a large number of different proteins. These so-called membrane proteins usually span the lipid matrix and protrude out into the space on either side of the membrane.

Over millions of years of evolution, cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. Our understanding of these machines and the insertion processes they mediate has reached a point where we have a very good picture of membrane protein biogenesis in various types of cells. However, more data still needs to be collected to completely comprehend the complex molecular mechanisms and the physical chemistry that underlies the different insertion processes.

The work presented in this thesis contributes to that understanding. More precisely, we have studied how weakly hydrophobic transmembrane elements of membrane proteins, which cannot spontaneously enter the largely apolar membrane matrices, are efficiently incorporated. Indeed, such elements are quite common in membrane proteins and our work has lead to the formulation of a novel mechanism for how they can be integrated into biological membranes.

We have also added to the understanding of the physical chemistry that underlies the membrane insertion process by systematically introducing non-proteinogenic amino acids into a membrane-spanning segment of a membrane protein and studying its membrane insertion capability.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2015. , 69 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-113384ISBN: 978-91-7649-089-1 (print)OAI: oai:DiVA.org:su-113384DiVA: diva2:784704
Public defence
2015-03-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2015-02-15 Created: 2015-01-29 Last updated: 2015-03-11Bibliographically approved
List of papers
1. Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
Open this publication in new window or tab >>Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
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2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, 221-229 p.Article in journal (Refereed) Published
Abstract [en]

In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

Keyword
marginally hydrophobic helix, membrane protein, topology, hydrophobicity
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry with Emphasis on Theoretical Chemistry
Identifiers
urn:nbn:se:su:diva-33926 (URN)10.1016/j.jmb.2009.11.036 (DOI)000274766500018 ()19931281 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 503265; 512092; 201924Swedish Research CouncilSwedish Foundation for Strategic Research
Note

authorCount :12

Available from: 2009-12-30 Created: 2009-12-30 Last updated: 2017-12-12Bibliographically approved
2. Orientational Preferences of Neighboring Helices Can Drive ER Insertion of a Marginally Hydrophobic Transmembrane Helix
Open this publication in new window or tab >>Orientational Preferences of Neighboring Helices Can Drive ER Insertion of a Marginally Hydrophobic Transmembrane Helix
2012 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 45, no 4, 529-540 p.Article in journal (Refereed) Published
Abstract [en]

alpha-helical integral membrane proteins critically depend on the correct insertion of their transmembrane alpha helices into the lipid bilayer for proper folding, yet a surprisingly large fraction of the transmembrane alpha helices in multispanning integral membrane proteins are not sufficiently hydrophobic to insert into the target membrane by themselves. How can such marginally hydrophobic segments nevertheless form transmembrane helices in the folded structure? Here, we show that a transmembrane helix with a strong orientational preference (N-cyt-C-lum or N-lum-C-cyt) can both increase and decrease the hydrophobicity threshold for membrane insertion of a neighboring, marginally hydrophobic helix. This effect helps explain the missing hydrophobicity in polytopic membrane proteins.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-76981 (URN)10.1016/j.molcel.2011.12.024 (DOI)000300753800012 ()
Note

4

Available from: 2012-06-05 Created: 2012-05-28 Last updated: 2017-12-07Bibliographically approved
3. Quantitative Analysis of SecYEG-Mediated Insertion of Transmembrane alpha-Helices into the Bacterial Inner Membrane
Open this publication in new window or tab >>Quantitative Analysis of SecYEG-Mediated Insertion of Transmembrane alpha-Helices into the Bacterial Inner Membrane
2013 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 425, no 15, 2813-2822 p.Article in journal (Refereed) Published
Abstract [en]

Most integral membrane proteins, both in prokaryotic and eukaryotic cells, are co-translationally inserted into the membrane via Sec-type translocons: the SecYEG complex in prokaryotes and the Sec61 complex in eukaryotes. The contributions of individual amino acids to the overall free energy of membrane insertion of single transmembrane alpha-helices have been measured for Sec61-mediated insertion into the endoplasmic reticulum (ER) membrane (Nature 450:1026-1030) but have not been systematically determined for SecYEG-mediated insertion into the bacterial inner membrane. We now report such measurements, carried out in Escherichia coli. Overall, there is a good correlation between the results found for the mammalian ER and the E. coli inner membrane, but the hydrophobicity threshold for SecYEG-mediated insertion is distinctly lower than that for Sec61-mediated insertion.

Keyword
SecYEG, leader peptidase, membrane protein, transmembrane helix
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-93567 (URN)10.1016/j.jmb.2013.04.025 (DOI)000322296400015 ()
Note

AuthorCount:4;

Available from: 2013-09-11 Created: 2013-09-10 Last updated: 2017-12-06Bibliographically approved
4. Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum
Open this publication in new window or tab >>Apolar surface area determines the efficiency of translocon-mediated membrane-protein integration into the endoplasmic reticulum
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2011 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 31, E359-E364 p.Article in journal (Refereed) Published
Abstract [en]

Integral membrane proteins are integrated cotranslationally into the membrane of the endoplasmic reticulum in a process mediated by the Sec61 translocon. Transmembrane α-helices in a translocating polypeptide chain gain access to the surrounding membrane through a lateral gate in the wall of the translocon channel [van den Berg B, et al. (2004) Nature427:36–44; Zimmer J, et al. (2008) Nature455:936–943; Egea PF, Stroud RM (2010)Proc Natl Acad Sci USA 107:17182–17187]. To clarify the nature of the membrane-integration process, we have measured the insertion efficiency into the endoplasmic reticulum membrane of model hydrophobic segments containing nonproteinogenic aliphatic and aromatic amino acids. We find that an amino acid’s contribution to the apparent free energy of membrane-insertion is directly proportional to the nonpolar accessible surface area of its side chain, as expected for thermodynamic partitioning between aqueous and nonpolar phases. But unlike bulk-phase partitioning, characterized by a nonpolar solvation parameter of 23 cal∕ðmol · Å2Þ, the solvation parameter for transfer from translocon to bilayer is 6 –10 cal∕ðmol · Å2Þ, pointing to important differences between translocon-guided partitioning and simple water-to-membrane partitioning. Our results provide compelling evidence for a termodynamic partitioning model and insights into the physical properties of the translocon.

Keyword
flexizyme, hydrophobicity, nonproteinogenic amino acid
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology; Biochemistry
Identifiers
urn:nbn:se:su:diva-71400 (URN)10.1073/pnas.1100120108 (DOI)000293385700008 ()21606334 (PubMedID)
Funder
Swedish Research Council
Available from: 2012-01-27 Created: 2012-01-27 Last updated: 2017-12-08Bibliographically approved

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