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Hydrophobic Clusters Raise the Threshold Hydrophilicity for Insertion of Transmembrane Sequences in Vivo
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).
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Number of Authors: 52016 (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.

Place, publisher, year, edition, pages
2016. Vol. 55, no 40, p. 5772-5779
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-136147DOI: 10.1021/acs.biochem.6b00650ISI: 000385336200013PubMedID: 27620701OAI: oai:DiVA.org:su-136147DiVA, id: diva2:1050792
Available from: 2016-11-30 Created: 2016-11-29 Last updated: 2017-09-14Bibliographically approved
In thesis
1. Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
Open this publication in new window or tab >>Insertion studies of model transmembrane segments into bacterial and eukaryotic membranes
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
Keywords
ribosome, membrane integration, translocation, arrest peptide, SecM, Xbp1
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-146869 (URN)978-91-7797-002-6 (ISBN)978-91-7797-003-3 (ISBN)
Public defence
2017-10-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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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

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