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Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
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.
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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.

Place, publisher, year, edition, pages
2010. Vol. 396, no 1, 221-229 p.
Keyword [en]
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: urn:nbn:se:su:diva-33926DOI: 10.1016/j.jmb.2009.11.036ISI: 000274766500018PubMedID: 19931281OAI: oai:DiVA.org:su-33926DiVA: diva2:283817
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
In thesis
1. Intra- and intermolecular interactions in proteins: Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.
Open this publication in new window or tab >>Intra- and intermolecular interactions in proteins: Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most of the processes in a living cell are carried out by proteins. Depending on the needs of the cell, different proteins will interact and form the molecular machines demanded for the moment. A subset of proteins called integral membrane proteins are responsible for the interchange of matter and information across the biological membrane, the lipid bilayer enveloping and defining the cell. Most of these proteins are co-translationally integrated into the membrane by the Sec translocation machinery.

This thesis addresses two questions that have emerged during the last decade. The first concerns membrane proteins: a number of α-helices have been observed to span the membrane in the obtained three-dimensional structures even though these helices are predicted not to be hydrophobic enough to be recognized by the translocon for integration. We show for a number of these marginally hydrophobic protein segments that they indeed do not insert well outside of their native context, but that their local sequence context can improve the level of integration mediated by the translocon. We also find that many of these helices are overlapped by more hydrophobic segments. We propose, supported by experimental results, that the latter are initially integrated into the membrane, followed by post-translational structural rearrangements. Finally, we investigate whether the integration of the marginally hydrophobic TMHs of the lactose permease of Escherichia coli is facilitated by the formation of hairpin structures. However our combined efforts of computational simulations and experimental investigations find no evidence for this.

The second question addressed in this thesis is that of the interpretation of the large datasets on which proteins that interact with each other in a cell. We have analyzed the results from several large-scale investigations concerning protein interactions in yeast and draw conclusions regarding the biases, strengths and weaknesses of these datasets and the methods used to obtain them.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholms universitet, 2010. 74 p.
Keyword
membrane proteins, membrane insertion, marginally hydrophobic helix, hydrophobicity, protein-protein interactions, LacY, GltPh
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42856 (URN)978-91-7447-111-3 (ISBN)
Public defence
2010-10-15, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense the following publications were not published and had a status as follows: Paper 2: In press; Paper 4 Manuscript.Available from: 2010-09-23 Created: 2010-09-16 Last updated: 2014-11-10Bibliographically approved
2. Membrane protein topogenesis
Open this publication in new window or tab >>Membrane protein topogenesis
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:nbn:se:su:diva-113384 (URN)978-91-7649-089-1 (ISBN)
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

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