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Dynamics of peptide chains during co-translational translocation, membrane integration & domain folding
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
ribosome, membrane integration, translocation, folding, arrest peptide, SecM
National Category
Biochemistry and Molecular Biology Cell Biology Biophysics
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-121764ISBN: 978-91-7649-285-7 (print)OAI: oai:DiVA.org:su-121764DiVA: diva2:861215
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
List of papers
1. 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, 1018-1022 p.Article 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
2. Charge-driven dynamics of nascent-chain movement through the SecYEG translocon
Open this publication in new window or tab >>Charge-driven dynamics of nascent-chain movement through the SecYEG translocon
2015 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, no 2, 145-149 p.Article in journal (Refereed) Published
Abstract [en]

On average, every fifth residue in secretory proteins carries either a positive or a negative charge. In a bacterium such as Escherichia coli, charged residues are exposed to an electric field as they transit through the inner membrane, and this should generate a fluctuating electric force on a translocating nascent chain. Here, we have used translational arrest peptides as in vivo force sensors to measure this electric force during cotranslational chain translocation through the SecYEG translocon. We find that charged residues experience a biphasic electric force as they move across the membrane, including an early component with a maximum when they are 47-49 residues away from the ribosomal P site, followed by a more slowly varying component. The early component is generated by the transmembrane electric potential, whereas the second may reflect interactions between charged residues and the periplasmic membrane surface.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-115293 (URN)10.1038/nsmb.2940 (DOI)000348967400010 ()25558985 (PubMedID)
Note

AuthorCount:4;

Available from: 2015-03-31 Created: 2015-03-18 Last updated: 2017-12-04Bibliographically approved
3. Cotranslational Protein Folding inside the Ribosome Exit Tunnel
Open this publication in new window or tab >>Cotranslational Protein Folding inside the Ribosome Exit Tunnel
Show others...
2015 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 12, no 10, 1533-1540 p.Article in journal (Refereed) Published
Abstract [en]

At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of co-translational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

Keyword
Ribosome, Protein Folding, Cotranslational Folding, Translation, SecM, Cryo-EM
National Category
Biochemistry and Molecular Biology Cell Biology Biophysics
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-121762 (URN)10.1016/j.celrep.2015.07.065 (DOI)000360965500002 ()
Available from: 2015-10-15 Created: 2015-10-15 Last updated: 2017-12-01Bibliographically approved
4. Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants
Open this publication in new window or tab >>Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants
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.

Keyword
Protein Secretion, Ribosome Function, Translation, Translation Elongation Factor, Translation Regulation, SecM
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-117450 (URN)10.1074/jbc.M115.641555 (DOI)000353241100027 ()25713070 (PubMedID)
Note

AuthorCount:4;

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2017-12-04Bibliographically approved

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