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Intra- and intermolecular interactions in proteins: Studies of marginally hydrophobic transmembrane alpha-helices and protein-protein interactions.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Elofsson)
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 [en]
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: urn:nbn:se:su:diva-42856ISBN: 978-91-7447-111-3 (print)OAI: oai:DiVA.org:su-42856DiVA: diva2:351779
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
List of papers
1. Quantitative assessment of the structural bias in protein-protein interaction assays.
Open this publication in new window or tab >>Quantitative assessment of the structural bias in protein-protein interaction assays.
2008 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 22, 4657-46667 p.Article in journal (Refereed) Published
Abstract [en]

With recent publications of several large-scale protein-protein interaction (PPI) studies, the realization of the full yeast interaction network is getting closer. Here, we have analysed several yeast protein interaction datasets to understand their strengths and weaknesses. In particular, we investigate the effect of experimental biases on some of the protein properties suggested to be enriched in highly connected proteins. Finally, we use support vector machines (SVM) to assess the contribution of these properties to protein interactivity. We find that protein abundance is the most important factor for detecting interactions in tandem affinity purifications (TAP), while it is of less importance for Yeast Two Hybrid (Y2H) screens. Consequently, sequence conservation and/or essentiality of hubs may be related to their high abundance. Further, proteins with disordered structure are over-represented in Y2H screens and in one, but not the other, large-scale TAP assay. Hence, disordered regions may be important both in transient interactions and interactions in complexes. Finally, a few domain families seem to be responsible for a large part of all interactions. Most importantly, we show that there are method-specific biases in PPI experiments. Thus, care should be taken before drawing strong conclusions based on a single dataset.

Place, publisher, year, edition, pages
Wiley, 2008
Keyword
abundance, disorder, protein-protein interactions
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-14849 (URN)000261380300007 ()18924110 (PubMedID)
Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2014-11-10Bibliographically approved
2. 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
Show others...
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: 2016-02-23Bibliographically approved
3. Repositioning of transmembrane alpha-helices during membrane protein folding
Open this publication in new window or tab >>Repositioning of transmembrane alpha-helices during membrane protein folding
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2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 397, no 1, 190-201 p.Article in journal (Refereed) Published
Abstract [en]

We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii Glt(Ph) glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified.

Keyword
membrane protein; GltPh; protein folding; membrane insertion
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42790 (URN)10.1016/j.jmb.2010.01.042 (DOI)000275785600013 ()20109468 (PubMedID)
Available from: 2010-09-16 Created: 2010-09-14 Last updated: 2014-11-10Bibliographically approved
4. Insertion Properties of Marginally Hydrophobic Helices in the LacY Lactose Permease Transporter
Open this publication in new window or tab >>Insertion Properties of Marginally Hydrophobic Helices in the LacY Lactose Permease Transporter
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Transmembrane helices are generally believed to be recognized individually by the translocon based on theirhydrophobicity, but it has been proposed that they could also be recognized as pairs of helices. The fact thatmost transmembrane helices are individually clearly hydrophobic seems to support separate helix insertion,but there are important exceptions where the helices are only borderline hydrophilic, at least according tosequence-based prediction. Conrming these patterns and characterizing their role for insertion of helices isan important part in deciphering membrane protein insertion and folding. Here, we use a combination ofsequence bioinformatics, simplied physical modeling, and experiments to investigate whether helices in theLacY lactose permease transporter are recognized by the translocon, and if not whether helix-helix interactionsmight stabilize their insertion. From the experimentally determined biological hydrophobicity scale, ve out of thetwelve transmembrane segments of LacY are predicted to have low spontaneous insertion, which is qualitativelyconrmed in a simplied simulation model using an implicit membrane environment as well as experimentallyin vitro. For some pairs a small, but signicant, increase in insertion eciency was seen both in the simulationsand in the in vitro system. However, the overall insertion eciency is only marginally increased when pairsof borderline hydrophobic helices are co-inserted, which suggests that translocon-mediated membrane insertionpredominantly recognizes individual helices. It also seems to imply that stabilization of marginally hydrophobichelices - at least for LacY - is a collective eect in the nal folded membrane protein, rather than caused by favorable interactions and hairpin formation during insertion.

Keyword
Membrane protein, transmembrane helix, hydrophobic, insertion, interactions, Monte Carlo
National Category
Biochemistry and Molecular Biology Bioinformatics and Systems Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-42792 (URN)
Available from: 2010-09-16 Created: 2010-09-14 Last updated: 2014-11-10Bibliographically approved

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