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Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cells are encapsulated by a biological membrane in order to separate the cell interior from the surrounding environment. Different lipids and proteins compose the membrane and present a semi-permeable barrier for the diffusion of ions and molecules across the lipid bilayer. Membrane proteins also mediate the passage of signals between the interior and the exterior of the cell.   To ensure the proper functioning of membrane proteins, it is essential that nascent membrane proteins are correctly integrated into the lipid bilayer to be able to fold and oligomerize.  In this thesis, an engineered protein containing two natural transmembrane segments followed by an additional test segment, has been used as a model protein to study (i) sequence requirements for translocon-mediated insertion of the test segment, (ii) dynamics of nascent membrane proteins undergoing translocon-mediated insertion and (iii) to carry out an extensive mutagenesis scan to identify critical residues in the mammalian arrest peptide Xbp1 that enhances translational stalling in the ribosome. This provides a toolbox of arrest peptides with different stalling strengths that will be useful for force measurements on nascent protein chains.     

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry, Stockholm Universityand Biophysics, Stockholm University , 2017. , p. 88
Keyword [en]
ribosome, membrane integration, translocation, arrest peptide, SecM, Xbp1
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-146869ISBN: 978-91-7797-002-6 (print)ISBN: 978-91-7797-003-3 (electronic)OAI: oai:DiVA.org:su-146869DiVA, id: diva2:1141218
Public defence
2017-10-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2017-10-03 Created: 2017-09-14 Last updated: 2017-10-04Bibliographically approved
List of papers
1. Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences
Open this publication in new window or tab >>Hydrophobic Blocks Facilitate Lipid Compatibility and Translocon Recognition of Transmembrane Protein Sequences
2015 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 7, p. 1465-1473Article in journal (Refereed) Published
Abstract [en]

Biophysical hydrophobicity scales suggest that partitioning of a protein segment from an aqueous phase into a membrane is governed by its perceived segmental hydrophobicity but do not establish specifically (i) how the segment is identified in vivo for translocon-mediated insertion or (ii) whether the destination lipid bilayer is biochemically receptive to the inserted sequence. To examine the congruence between these dual requirements, we designed and synthesized a library of Lys-tagged peptides of a core length sufficient to span a bilayer but with varying patterns of sequence, each composed of nine Leu residues, nine Ser residues, and one (central) Trp residue. We found that peptides containing contiguous Leu residues (Leu-block peptides, e.g., LLLLLLLLLWSSSSSSSSS), in comparison to those containing discontinuous stretches of Leu residues (non-Leu-block peptides, e.g., SLSLLSLSSWSLLSLSLLS), displayed greater helicity (circular dichroism spectroscopy), traveled slower during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had longer reverse phase high-performance liquid chromatography retention times on a C-18 column, and were helical when reconstituted into 1-palmitoyl-2-oleoylglycero-3-phosphocholine liposomes, each observation indicating superior lipid compatibility when a Leu-block is present. These parameters were largely paralleled in a biological membrane insertion assay using microsomal membranes from dog pancreas endoplasmic reticulum, where we found only the Leu-block sequences successfully inserted; intriguingly, an amphipathic peptide (SLLSSLLSSWLLSSLLSSL; Leu face, Ser face) with biophysical properties similar to those of Leu-block peptides failed to insert. Our overall results identify local sequence lipid compatibility rather than average hydrophobicity as a principal determinant of transmembrane segment potential, while demonstrating that further subtleties of hydrophobic and helical patterning, such as circumferential hydrophobicity in Leu-block segments, promote translocon-mediated insertion.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-115912 (URN)10.1021/bi5014886 (DOI)000350193800004 ()25635746 (PubMedID)
Note

AuthorCount:4;

Available from: 2015-04-17 Created: 2015-04-08 Last updated: 2017-09-14Bibliographically approved
2. Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo
Open this publication in new window or tab >>Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo
Show others...
2016 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 40, p. 5772-5779Article in journal (Refereed) Published
Abstract [en]

Insertion of a nascent membrane protein segment by the translocon channel into the bilayer is naturally promoted by high segmental hydrophobicity, but its selection as a transmembrane (TM) segment is complicated by the diverse environments (aqueous vs lipidic) the protein encounters and by the fact that most TM segments contain a substantial amount (similar to 30%) of polar residues, as required for protein structural stabilization and/or function. To examine the contributions of these factors systematically, we designed and synthesized a peptide library consisting of pairs of compositionally identical, but sequentially different, peptides with 19-residue core sequences varying (i) in Leu positioning (with five or seven Leu residues clustered into a contiguous block in the middle of the segment or scrambled throughout the sequence) and (ii) in Ser content (0-6 residues). The library was analyzed by a combination of biophysical and biological techniques, including HPLC retention times, circular dichroism measurements of helicity in micelle and phospholipid bilayer media, and relative blue shifts in Trp fluorescence maxima, as well as by the extent of membrane insertion in a translocon-mediated assay using microsomal membranes from dog pancreas endoplasmic reticulum. We found that local blocks of high hydrophobicity heighten the translocon's propensity to insert moderately hydrophilic sequences, until a threshold hydrophilicity is surpassed whereby segments no longer insert even in the presence of Leu blocks. This study codifies the prerequisites of apolar/polar content and residue positioning that define nascent TM segments, illustrates the accuracy in their prediction, and highlights how a single disease-causing mutation can tip the balance toward anomaloug translocation/insertion.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-136147 (URN)10.1021/acs.biochem.6b00650 (DOI)000385336200013 ()27620701 (PubMedID)
Available from: 2016-11-30 Created: 2016-11-29 Last updated: 2017-09-14Bibliographically approved
3. A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration
Open this publication in new window or tab >>A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration
2012 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 19, no 10, p. 1018-1022Article in journal (Refereed) Published
Abstract [en]

Membrane proteins destined for insertion into the inner membrane of bacteria or the endoplasmic reticulum membrane in eukaryotic cells are synthesized by ribosomes bound to the bacterial SecYEG or the homologous eukaryotic Sec61 translocon. During co-translational membrane integration, transmembrane alpha-helical segments in the nascent chain exit the translocon through a lateral gate that opens toward the surrounding membrane, but the mechanism of lateral exit is not well understood. In particular, little is known about how a transmembrane helix behaves when entering and exiting the translocon. Using translation-arrest peptides from bacterial SecM proteins and from the mammalian Xbp1 protein as force sensors, we show that substantial force is exerted on a transmembrane helix at two distinct points during its transit through the translocon channel, providing direct insight into the dynamics of membrane integration.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-82433 (URN)10.1038/nsmb.2376 (DOI)000309591000010 ()
Funder
EU, European Research Council, ERC-2008-AdG 232648
Note

AuthorCount:4;

Available from: 2012-11-14 Created: 2012-11-14 Last updated: 2017-09-14Bibliographically approved
4. Mutational analysis of the human Xbp1 translational arrest peptide and construction of arrest-enhanced variants
Open this publication in new window or tab >>Mutational analysis of the human Xbp1 translational arrest peptide and construction of arrest-enhanced variants
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Xbp1, a protein involved in the unfolded protein response, is a rare example of a mammalian protein that contains a well-defined translational arrest peptide (AP). In order to define the critical residues in the Xbp1u AP, and to search for variants with stronger arrest potency than the wildtype Xbp1u AP, we have carried out a full mutagenesis scan where each residue in the AP was replaced by the other 19 natural amino acids. We find that 10 of the 21 mutagenized positions are optimal already in the wildtype Xbp1 AP, while certain mutations in the remaining residues lead to a strong increase in the arrest potency. Xbp1 has thus evolved to induce an intermediate level of translational arrest, and versions with much stronger arrest efficiency exist. We further show Xbp1- induced translational arrest is reduced in response to increased tension in the nascent chain, making it possible to carry out studies in mammalian systems of cotranslational processes such as membrane protein assembly and protein folding by using suitable Xbp1 AP variants as “force sensors”, as has been done previously in E. coli using bacterial APs.

Keyword
Arrest peptide, translation, Xbp1
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-147391 (URN)
Available from: 2017-09-26 Created: 2017-09-26 Last updated: 2017-09-26Bibliographically approved

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