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Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 16, 10208-10215 p.Article in journal (Refereed) Published
Abstract [en]

Translational arrest peptides (APs) are short stretches of polypeptides that induce translational stalling when synthesized on a ribosome. Mechanical pulling forces acting on the nascent chain can weaken or even abolish stalling. APs can therefore be used as in vivo force sensors, making it possible to measure the forces that act on a nascent chain during translation with single-residue resolution. It is also possible to score the relative strengths of APs by subjecting them to a given pulling force and ranking them according to stalling efficiency. Using the latter approach, we now report an extensive mutagenesis scan of a strong mutant variant of the Mannheimia succiniciproducens SecM AP and identify mutations that further increase the stalling efficiency. Combining three such mutations, we designed an AP that withstands the strongest pulling force we are able to generate at present. We further show that diproline stretches in a nascent protein act as very strong APs when translation is carried out in the absence of elongation factor P. Our findings highlight critical residues in APs, show that certain amino acid sequences induce very strong translational arrest and provide a toolbox of APs of varying strengths that can be used for in vivo force measurements.

Place, publisher, year, edition, pages
2015. Vol. 290, no 16, 10208-10215 p.
Keyword [en]
Protein Secretion, Ribosome Function, Translation, Translation Elongation Factor, Translation Regulation, SecM
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-117450DOI: 10.1074/jbc.M115.641555ISI: 000353241100027PubMedID: 25713070OAI: oai:DiVA.org:su-117450DiVA: diva2:812557
Note

AuthorCount:4;

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding
Open this publication in new window or tab >>Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts.

The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2015. 50 p.
Keyword
ribosome, membrane integration, translocation, folding, arrest peptide, SecM
National Category
Biochemistry and Molecular Biology Cell Biology Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-121764 (URN)978-91-7649-285-7 (ISBN)
Public defence
2015-12-04, Magnéli Hall, Arrhenius Laboratory, Svante arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2015-11-12 Created: 2015-10-15 Last updated: 2015-11-03Bibliographically approved

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Cymer, FlorianHedman, RickardIsmail, Nurzianvon Heijne, Gunnar
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