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The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center (SeRC), Sweden.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2014 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 16, 2982-2991 p.Article in journal (Refereed) Published
Abstract [en]

The translocon recognizes transmembrane helices with sufficient level of hydrophobicity and inserts them into the membrane. However, sometimes less hydrophobic helices are also recognized. Positive inside rule, orientational preferences of and specific interactions with neighboring helices have been shown to aid in the recognition of these helices, at least in artificial systems. To better understand how the translocon inserts marginally hydrophobic helices, we studied three naturally occurring marginally hydrophobic helices, which were previously shown to require the subsequent helix for efficient translocon recognition. We find no evidence for specific interactions when we scan all residues in the subsequent helices. Instead, we identify arginines located at the N-terminal part of the subsequent helices that are crucial for the recognition of the marginally hydrophobic transmembrane helices, indicating that the positive inside rule is important. However, in two of the constructs, these arginines do not aid in the recognition without the rest of the subsequent helix; that is, the positive inside rule alone is not sufficient. Instead, the improved recognition of marginally hydrophobic helices can here be explained as follows: the positive inside rule provides an orientational preference of the subsequent helix, which in turn allows the marginally hydrophobic helix to be inserted; that is, the effect of the positive inside rule is stronger if positively charged residues are followed by a transmembrane helix. Such a mechanism obviously cannot aid C-terminal helices, and consequently, we find that the terminal helices in multi-spanning membrane proteins are more hydrophobic than internal helices.

Place, publisher, year, edition, pages
2014. Vol. 426, no 16, 2982-2991 p.
Keyword [en]
marginally hydrophobic helices, translocon recognition, membrane proteins, positive inside rule, orientational preference
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-107079DOI: 10.1016/j.jmb.2014.06.002ISI: 000340327500007OAI: oai:DiVA.org:su-107079DiVA: diva2:743090
Note

AuthorCount:7;

Available from: 2014-09-03 Created: 2014-09-03 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding
Open this publication in new window or tab >>Marginally hydrophobic transmembrane α-helices shaping membrane protein folding
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most membrane proteins are inserted into the membrane co-translationally utilizing the translocon, which allows a sufficiently long and hydrophobic stretch of amino acids to partition into the membrane. However, X-ray structures of membrane proteins have revealed that some transmembrane helices (TMHs) are surprisingly hydrophilic. These marginally hydrophobic transmembrane helices (mTMH) are not recognized as TMHs by the translocon in the absence of local sequence context.

We have studied three native mTMHs, which were previously shown to depend on a subsequent TMH for membrane insertion. Their recognition was not due to specific interactions. Instead, the presence of basic amino acids in their cytoplasmic loop allowed membrane insertion of one of them. In the other two, basic residues are not sufficient unless followed by another, hydrophobic TMH. Post-insertional repositioning are another way to bring hydrophilic residues into the membrane. We show how four long TMHs with hydrophilic residues seen in X-ray structures, are initially inserted as much shorter membrane-embedded segments. Tilting is thus induced after membrane-insertion, probably through tertiary packing interactions within the protein.

Aquaporin 1 illustrates how a mTMH can shape membrane protein folding and how repositioning can be important in post-insertional folding. It initially adopts a four-helical intermediate, where mTMH2 and TMH4 are not inserted into the membrane. Consequently, TMH3 is inserted in an inverted orientation. The final conformation with six TMHs is formed by TMH2 and 4 entering the membrane and TMH3 rotating 180°. Based on experimental and computational results, we propose a mechanism for the initial step in the folding of AQP1: A shift of TMH3 out from membrane core allows the preceding regions to enter the membrane, which provides flexibility for TMH3 to re-insert in its correct orientation.

Place, publisher, year, edition, pages
Stockholm: Department of biochemistry and biophysics, Stockholm University, 2014. 66 p.
Keyword
membrane protein folding, hydrophobicity, translocon, transmembrane helix, marginally hydrophobic transmembrane helices, orientational preference, positive inside rule, aquaporin 1
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-109335 (URN)978-91-7649-050-1 (ISBN)
Public defence
2014-12-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrheniusväg 16 B, Stockholm, 13:00 (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: 2014-11-27 Created: 2014-11-18 Last updated: 2014-11-28Bibliographically approved
2. Topology Prediction of α-Helical Transmembrane Proteins
Open this publication in new window or tab >>Topology Prediction of α-Helical Transmembrane Proteins
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins fulfil a number of tasks in cells, including signalling, cell-cell interaction, and the transportation of molecules. The prominence of these tasks makes membrane proteins an important target for clinical drugs. Because of the decreasing price of sequencing, the number of sequences known is increasing at such a rate that manual annotations cannot compete. Here, topology prediction is a way to provide additional information. It predicts the location and number of transmembrane helices in the protein and the orientation inside the membrane. An important factor to detect transmembrane helices is their hydrophobicity, which can be calculated using dedicated scales. In the first paper, we studied the difference between several hydrophobicity scales and evaluated their performance. We showed that while they appear to be similar, their performance for topology prediction differs significantly. The better performing scales appear to measure the probability of amino acids to be within a transmembrane helix, instead of just being located in a hydrophobic environment.

Around 20% of the transmembrane helices are too hydrophilic to explain their insertion with hydrophobicity alone. These are referred to as marginally hydrophobic helices. In the second paper, we studied three of these helices experimentally and performed an analysis on membrane proteins. The experiments show that for all three helices positive charges on the N-terminal side of the subsequent helix are important to insert, but only two need the subsequent helix. Additionally, the analysis shows that not only the N-terminal helices are more hydrophobic, but also the C-terminal transmembrane helices.

In Paper III, the finding from the second paper was used to improve the topology prediction. By extending our hidden Markov model with N- and C-terminal helix states, we were able to set stricter cut-offs. This improved the general topology prediction and in particular miss-prediction in large N- and C-terminal domains, as well the separation between transmembrane and non-transmembrane proteins.

Lastly, we contribute several new features to our consensus topology predictor, TOPCONS. We added states for the detection of signal peptides to its hidden Markov model and thus reduce the over-prediction of transmembrane helices. With a new method for the generation of profile files, it is possible to increase the size of the database used to find homologous proteins and decrease the running time by 75%.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2016. 46 p.
National Category
Bioinformatics (Computational Biology)
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-129061 (URN)
Public defence
2016-06-03, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-05-11 Created: 2016-04-13 Last updated: 2017-02-24Bibliographically approved

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