Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Marginally hydrophobic transmembrane α-helices shaping membrane protein folding
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Arne Elofsson)
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 [en]
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: urn:nbn:se:su:diva-109335ISBN: 978-91-7649-050-1 (print)OAI: oai:DiVA.org:su-109335DiVA: diva2:764217
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
List of papers
1. The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix
Open this publication in new window or tab >>The Positive Inside Rule Is Stronger When Followed by a Transmembrane Helix
Show others...
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.

Keyword
marginally hydrophobic helices, translocon recognition, membrane proteins, positive inside rule, orientational preference
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-107079 (URN)10.1016/j.jmb.2014.06.002 (DOI)000340327500007 ()
Note

AuthorCount:7;

Available from: 2014-09-03 Created: 2014-09-03 Last updated: 2017-12-05Bibliographically approved
2. Insertion of marginally hydrophobic helix in EmrD
Open this publication in new window or tab >>Insertion of marginally hydrophobic helix in EmrD
2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

The positive inside rule governs the orientation of membrane proteins in such a way that more positively chargedamino acids are found on the inside of a cell. The exact mechanisms of how this is achieved are not well known, butit is clear that positively charged residues can facilitate the insertion of transmembrane helices that are not sufficientlyhydrophobic to be inserted otherwise. Here, we study one such helix, helix 2 in EmrD. We show that the insertionof this helix is facilitated when followed by positively charged residues and a hydrophobic helix. Surprisingly thepositively charged residues are not sufficient alone. This further strengthens our earlier observations that the last helixneeds to be more hydrophobic than previous helices.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-108912 (URN)
Available from: 2014-11-17 Created: 2014-11-06 Last updated: 2016-05-27Bibliographically approved
3. Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation
Open this publication in new window or tab >>Large Tilts in Transmembrane Helices Can Be Induced during Tertiary Structure Formation
Show others...
2014 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, no 13, 2529-2538 p.Article in journal (Refereed) Published
Abstract [en]

While early structural models of helix-bundle integral membrane proteins posited that the transmembrane a-helices [transmembrane helices (TMHs)] were orientated more or less perpendicular to the membrane plane, there is now ample evidence from high-resolution structures that many TMHs have significant tilt angles relative to the membrane. Here, we address the question whether the tilt is an intrinsic property of the TMH in question or if it is imparted on the TMH during folding of the protein. Using a glycosylation mapping technique, we show that four highly tilted helices found in multi-spanning membrane proteins all have much shorter membrane-embedded segments when inserted by themselves into the membrane than seen in the high-resolution structures. This suggests that tilting can be induced by tertiary packing interactions within the protein, subsequent to the initial membrane-insertion step.

Keyword
transnnembrane helix, membrane protein folding, translocon
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-106063 (URN)10.1016/j.jmb.2014.04.020 (DOI)000337780200009 ()
Note

AuthorCount:9;

Available from: 2014-07-31 Created: 2014-07-21 Last updated: 2017-12-05Bibliographically approved
4. Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core
Open this publication in new window or tab >>Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core
Show others...
2014 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 23, no 7, 981-992 p.Article in journal (Refereed) Published
Abstract [en]

The folding of most integral membrane proteins follows a two-step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four-helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six-helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD-simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process.

Keyword
membrane protein, translocon recognition, protein folding, hydrophobicity, molecular dynamics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-106195 (URN)10.1002/pro.2483 (DOI)000337669800014 ()
Note

AuthorCount:6;

Available from: 2014-07-31 Created: 2014-07-28 Last updated: 2017-12-05Bibliographically approved

Open Access in DiVA

Marginally hydrophobic transmembrane alpha-helices shaping membrane protein folding(5699 kB)335 downloads
File information
File name FULLTEXT01.pdfFile size 5699 kBChecksum SHA-512
d47148f23319e59d59aad7d5f6dd00f000a5721222da6d787750dccd3d439d3ed5c99a38ddf6deb4775bd69f2112ae6577681ba8af0a1d02d5f4822f15bb7eb0
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
de Marothy, Tuuli Minttu Virkki
By organisation
Department of Biochemistry and Biophysics
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 335 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 714 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf