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Mermans, D., Nicolaus, F., Baygin, A. & von Heijne, G. (2023). Cotranslational folding of human growth hormone in vitro and in Escherichia coli. FEBS Letters, 597(10), 1355-1362
Open this publication in new window or tab >>Cotranslational folding of human growth hormone in vitro and in Escherichia coli
2023 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 597, no 10, p. 1355-1362Article in journal (Refereed) Published
Abstract [en]

Human growth hormone (hGH) is a four-helix bundle protein of considerable pharmacological interest. Recombinant hGH is produced in bacteria, yet little is known about its folding during expression in Escherichia coli. We have studied the cotranslational folding of hGH using force profile analysis (FPA), both during in vitro translation in the absence and presence of the chaperone trigger factor (TF), and when expressed in E. coli. We find that the main folding transition starts before hGH is completely released from the ribosome, and that it can interact with TF and possibly other chaperones. 

Keywords
cotranslational protein folding, human growth hormone
National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-213806 (URN)10.1002/1873-3468.14562 (DOI)000902903800001 ()36520514 (PubMedID)2-s2.0-85145052910 (Scopus ID)
Available from: 2023-01-25 Created: 2023-01-25 Last updated: 2023-10-09Bibliographically approved
White, S. H., von Heijne, G. & Engelman, D. M. (2022). Cell boundaries: How membranes and their proteins work. CRC Press
Open this publication in new window or tab >>Cell boundaries: How membranes and their proteins work
2022 (English)Book (Other academic)
Abstract [en]

The central themes of Cell Boundaries concern the structural and organizational principles underlying cell membranes, and how these principles enable function. By building a biological and biophysical foundation for understanding the organization of lipids in bilayers and the folding, assembly, stability, and function of membrane proteins, the book aims to broaden the knowledge of bioscience students to include the basic physics and physical chemistry that inform us about membranes. In doing so, it is hoped that physics students will find familiar territory that will lead them to an interest in biology. Our progress toward understanding membranes and membrane proteins depends strongly upon the concerted use of both biology and physics. It is important for students to know not only what we know, but how we have come to know it, so Cell Boundaries endeavours to bring out the history behind the central discoveries, especially in the early chapters, where the foundation is laid for later chapters. Science is far more interesting if, as students, we can appreciate and share in the adventures-and misadventures-of discovering new scientific knowledge. Cell Boundaries was written with advanced undergraduates and beginning graduate students in the biological and physical sciences in mind, though this textbook will likely have appeal to researchers and other academics as well. Highlights the history of important central discoveries Early chapters lay the foundation for later chapters to build on, so knowledge is amassed High-quality line diagrams illustrate key concepts and illuminate molecular mechanisms Box features and spreads expand on topics in main text, including histories of discoveries, special techniques, and applications. 

Place, publisher, year, edition, pages
CRC Press, 2022. p. 546
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-212281 (URN)10.1201/9780429341328 (DOI)2-s2.0-85126158202 (Scopus ID)9780815342168 (ISBN)9780429341328 (ISBN)
Available from: 2022-12-08 Created: 2022-12-08 Last updated: 2022-12-08Bibliographically approved
Mermans, D., Nicolaus, F., Fleisch, K. & von Heijne, G. (2022). Cotranslational folding and assembly of the dimeric Escherichia coli inner membrane protein EmrE. Proceedings of the National Academy of Sciences of the United States of America, 119(35), Article ID e2205810119.
Open this publication in new window or tab >>Cotranslational folding and assembly of the dimeric Escherichia coli inner membrane protein EmrE
2022 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 119, no 35, article id e2205810119Article in journal (Refereed) Published
Abstract [en]

In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)—a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion—to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process. 

Keywords
cotranslational dimerization, cotranslational folding, EmrE, membrane protein biogenesis, EmrE protein, membrane protein, monomer, polypeptide, unclassified drug, antiporter, EmrE protein, E coli, Escherichia coli protein, peptide, amino terminal sequence, Article, carboxy terminal sequence, dimerization, Escherichia coli, nonhuman, polyacrylamide gel electrophoresis, protein assembly, protein engineering, protein folding, protein interaction, protein structure, ribosome, genetics, metabolism, protein synthesis, Antiporters, Escherichia coli Proteins, Membrane Proteins, Peptides, Protein Biosynthesis
National Category
Biophysics
Identifiers
urn:nbn:se:su:diva-212052 (URN)10.1073/pnas.2205810119 (DOI)000911585800023 ()35994672 (PubMedID)2-s2.0-85136169899 (Scopus ID)
Available from: 2022-12-01 Created: 2022-12-01 Last updated: 2023-02-14Bibliographically approved
Di Palma, F., Decherchi, S., Pardo-Avila, F., Succi, S., Levitt, M., von Heijne, G. & Cavalli, A. (2022). Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome. Journal of Chemical Theory and Computation, 18(3), 1905-1914
Open this publication in new window or tab >>Probing Interplays between Human XBP1u Translational Arrest Peptide and 80S Ribosome
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2022 (English)In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 18, no 3, p. 1905-1914Article in journal (Refereed) Published
Abstract [en]

The ribosome stalling mechanism is a crucial biological process, yet its atomistic underpinning is still elusive. In this framework, the human XBP1u translational arrest peptide (AP) plays a central role in regulating the unfolded protein response (UPR) in eukaryotic cells. Here, we report multimicrosecond all-atom molecular dynamics simulations designed to probe the interactions between the XBP1u AP and the mammalian ribosome exit tunnel, both for the wild type AP and for four mutant variants of different arrest potencies. Enhanced sampling simulations allow investigating the AP release process of the different variants, shedding light on this complex mechanism. The present outcomes are in qualitative/quantitative agreement with available experimental data. In conclusion, we provide an unprecedented atomistic picture of this biological process and clear-cut insights into the key AP-ribosome interactions.

Keywords
Peptides and proteins, Genetics, Chemical structure, Molecular interactions, Extraction
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-200562 (URN)10.1021/acs.jctc.1c00796 (DOI)000812201900001 ()34881571 (PubMedID)2-s2.0-85121231775 (Scopus ID)
Available from: 2022-01-07 Created: 2022-01-07 Last updated: 2022-06-28Bibliographically approved
Teufel, F., Almagro Armenteros, J. J., Rosenberg Johansen, A., Gíslason, M. H., Pihl, S. I., Tsirigos, K. D., . . . Nielsen, H. (2022). SignalP 6.0 predicts all five types of signal peptides using protein language models. Nature Biotechnology, 40(7), 1023-1025
Open this publication in new window or tab >>SignalP 6.0 predicts all five types of signal peptides using protein language models
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2022 (English)In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 40, no 7, p. 1023-1025Article in journal (Refereed) Published
Abstract [en]

Signal peptides (SPs) are short amino acid sequences that control protein secretion and translocation in all living organisms. SPs can be predicted from sequence data, but existing algorithms are unable to detect all known types of SPs. We introduce SignalP 6.0, a machine learning model that detects all five SP types and is applicable to metagenomic data. A new version of SignalP predicts all types of signal peptides.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-201108 (URN)10.1038/s41587-021-01156-3 (DOI)000737730200002 ()34980915 (PubMedID)2-s2.0-85122179157 (Scopus ID)
Available from: 2022-01-21 Created: 2022-01-21 Last updated: 2022-08-04Bibliographically approved
Nicolaus, F., Ibrahimi, F., den Besten, A. & von Heijne, G. (2022). Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices. FEBS Letters, 596(8), 1004-1012
Open this publication in new window or tab >>Upstream charged and hydrophobic residues impact the timing of membrane insertion of transmembrane helices
2022 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 596, no 8, p. 1004-1012Article in journal (Refereed) Published
Abstract [en]

During SecYEG-mediated cotranslational insertion of membrane proteins, transmembrane helices (TMHs) first make contact with the membrane when their N-terminal end is ~ 45 residues away from the peptidyl transferase centre. However, we recently uncovered instances where the first contact is delayed by up to ~ 10 residues. Here, we recapitulate these effects using a model TMH fused to two short segments from the Escherichia coli inner membrane protein BtuC: a positively charged loop and a re-entrant loop. We show that the critical residues are two Arg residues in the positively charged loop and four hydrophobic residues in the re-entrant loop. Thus, both electrostatic and hydrophobic interactions involving sequence elements that are not part of a TMH can impact the way the latter behaves during membrane insertion. 

Keywords
BtuC, cotranslational, membrane protein biogenesis, transmembrane helix
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-201945 (URN)10.1002/1873-3468.14286 (DOI)000748497600001 ()35038773 (PubMedID)2-s2.0-85123923427 (Scopus ID)
Available from: 2022-02-09 Created: 2022-02-09 Last updated: 2022-06-09Bibliographically approved
Sandhu, H., Hedman, R., Cymer, F., Kudva, R., Ismail, N. & von Heijne, G. (2021). Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli. Journal of Molecular Biology, 433(15), Article ID 167047.
Open this publication in new window or tab >>Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli
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2021 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 433, no 15, article id 167047Article in journal (Refereed) Published
Abstract [en]

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) - a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide - to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB's two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the similar to 15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is similar to 70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Keywords
cotranslational protein folding, LepB, E. coli, periplasm
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-197124 (URN)10.1016/j.jmb.2021.167047 (DOI)000672680300008 ()33989648 (PubMedID)
Available from: 2021-09-28 Created: 2021-09-28 Last updated: 2022-02-25Bibliographically approved
von Heijne, G. (2021). Introduction to the Theme on Membrane Channels. Annual Review of Biochemistry, 90, 503-505
Open this publication in new window or tab >>Introduction to the Theme on Membrane Channels
2021 (English)In: Annual Review of Biochemistry, ISSN 0066-4154, E-ISSN 1545-4509, Vol. 90, p. 503-505Article, review/survey (Refereed) Published
Abstract [en]

This volume of the Annual Review of Biochemistry contains three reviews on membrane channel proteins: the first by Szczot et al., titled The Form and Function of PIEZO2; the second by Ruprecht & Kunji, titled Structural Mechanism of Transport of Mitochondrial Carriers; and the third by McIlwain et al., titled Membrane Exporters of Fluoride Ion. These reviews provide nice illustrations of just how far evolution has been able to play with the basic helix-bundle architecture of integral membrane proteins to produce membrane channels and transporters of widely different functions.

Keywords
membrane channels, PIEZO2, ADP/ATP carrier, fluoride channel, Fluc
National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-196424 (URN)10.1146/annurev-biochem-010421-023239 (DOI)000669645900019 ()34153216 (PubMedID)
Available from: 2021-09-08 Created: 2021-09-08 Last updated: 2022-02-25Bibliographically approved
Nicolaus, F., Metola, A., Mermans, D., Liljenström, A., Krč, A., Abdullahi, S. M., . . . von Heijne, G. (2021). Residue-by-residue analysis of cotranslational membrane protein integration in vivo. eLIFE, 10, Article ID e64302.
Open this publication in new window or tab >>Residue-by-residue analysis of cotranslational membrane protein integration in vivo
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2021 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 10, article id e64302Article in journal (Refereed) Published
Abstract [en]

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-192585 (URN)10.7554/eLife.64302 (DOI)000620792100001 ()33554862 (PubMedID)
Available from: 2021-04-26 Created: 2021-04-26 Last updated: 2022-04-19Bibliographically approved
Su, T., Kudva, R., Becker, T., Buschauer, R., Komar, T., Berninghausen, O., . . . Beckmann, R. (2021). Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide. Nucleic Acids Research, 49(16), 9539-9547
Open this publication in new window or tab >>Structural basis of L-tryptophan-dependent inhibition of release factor 2 by the TnaC arrest peptide
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2021 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 49, no 16, p. 9539-9547Article in journal (Refereed) Published
Abstract [en]

In Escherichia coli, elevated levels of free l-tryptophan (l-Trp) promote translational arrest of the TnaC peptide by inhibiting its termination. However, the mechanism by which translation-termination by the UGA-specific decoding release factor 2 (RF2) is inhibited at the UGA stop codon of stalled TnaC-ribosome-nascent chain complexes has so far been ambiguous. This study presents cryo-EM structures for ribosomes stalled by TnaC in the absence and presence of RF2 at average resolutions of 2.9 and 3.5 Å, respectively. Stalled TnaC assumes a distinct conformation composed of two small α-helices that act together with residues in the peptide exit tunnel (PET) to coordinate a single L-Trp molecule. In addition, while the peptidyl-transferase center (PTC) is locked in a conformation that allows RF2 to adopt its canonical position in the ribosome, it prevents the conserved and catalytically essential GGQ motif of RF2 from adopting its active conformation in the PTC. This explains how translation of the TnaC peptide effectively allows the ribosome to function as a L-Trp-specific small-molecule sensor that regulates the tnaCAB operon.

National Category
Biological Sciences
Identifiers
urn:nbn:se:su:diva-198856 (URN)10.1093/nar/gkab665 (DOI)000701664900039 ()34403461 (PubMedID)
Available from: 2021-11-17 Created: 2021-11-17 Last updated: 2021-11-17Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4490-8569

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