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  • 1. Almagro Armenteros, Jose Juan
    et al.
    Salvatore, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Emanuelsson, Olof
    Winther, Ole
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nielsen, Henrik
    Detecting Novel Sequence Signals in Targeting Peptides Using Deep LearningManuscript (preprint) (Other academic)
  • 2. Almagro Armenteros, José Juan
    et al.
    Tsirigos, Konstantinos D.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Technical University of Denmark, Denmark; Max Planck Institute for Molecular Genetics, Germany.
    Kaae Sonderby, Casper
    Nordahl Petersen, Thomas
    Winther, Ole
    Brunak, Søren
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nielsen, Henrik
    SignalP 5.0 improves signal peptide predictions using deep neural networks2019In: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 37, no 4, p. 420-423Article in journal (Refereed)
    Abstract [en]

    Signal peptides (SPs) are short amino acid sequences in the amino terminus of many newly synthesized proteins that target proteins into, or across, membranes. Bioinformatic tools can predict SPs from amino acid sequences, but most cannot distinguish between various types of signal peptides. We present a deep neural network-based approach that improves SP prediction across all domains of life and distinguishes between three types of prokaryotic SPs.

  • 3. Amico, Mauro
    et al.
    Finelli, Michele
    Rossi, Ivan
    Zauli, Andrea
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Viklund, Håkan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jones, David
    Krogh, Anders
    Fariselli, Piero
    Luigi Martelli, Pier
    Casadio, Rita
    PONGO: a web server for multiple predictions of all-alpha transmembrane proteins.2006In: Nucleic Acids Res, ISSN 1362-4962, Vol. 34, no Web Server issue, p. W169-72Article in journal (Refereed)
  • 4. Andersson, Helena
    et al.
    D'Antona, Aaron M
    Kendall, Debra A
    Von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chin, Chen-Ni
    Membrane assembly of the cannabinoid receptor 1: impact of a long N-terminal tail.2003In: Mol Pharmacol, ISSN 0026-895X, Vol. 64, no 3, p. 570-7Article in journal (Refereed)
  • 5. Baeza-Delgado, Carlos
    et al.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Marti-Renom, Marc A.
    Mingarro, Ismael
    Biological insertion of computationally designed short transmembrane segments2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 23397Article in journal (Refereed)
    Abstract [en]

    The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design.

  • 6. Bañó-Polo, Manuel
    et al.
    Baeza-Delgado, Carlos
    Tamborero, Silvia
    Hazel, Anthony
    Grau, Brayan
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Whitley, Paul
    Gumbart, James C.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mingarro, Ismael
    Transmembrane but not soluble helices fold inside the ribosome tunnel2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 5246Article in journal (Refereed)
    Abstract [en]

    Integral membrane proteins are assembled into the ER membrane via a continuous ribosome-translocon channel. The hydrophobicity and thickness of the core of the membrane bilayer leads to the expectation that transmembrane (TM) segments minimize the cost of harbouring polar polypeptide backbones by adopting a regular pattern of hydrogen bonds to form a-helices before integration. Co-translational folding of nascent chains into an a-helical conformation in the ribosomal tunnel has been demonstrated previously, but the features governing this folding are not well understood. In particular, little is known about what features influence the propensity to acquire a-helical structure in the ribosome. Using in vitro translation of truncated nascent chains trapped within the ribosome tunnel and molecular dynamics simulations, we show that folding in the ribosome is attained for TM helices but not for soluble helices, presumably facilitating SRP (signal recognition particle) recognition and/or a favourable conformation for membrane integration upon translocon entry.

  • 7. Bendtsen, Jannick Dyrløv
    et al.
    Jensen, Lars Juhl
    Blom, Nikolaj
    Von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brunak, Søren
    Feature-based prediction of non-classical and leaderless protein secretion.2004In: Protein Eng Des Sel, ISSN 1741-0126, Vol. 17, no 4, p. 349-56Article in journal (Refereed)
  • 8. Bendtsen, Jannick Dyrløv
    et al.
    Nielsen, Henrik
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brunak, Søren
    Improved prediction of signal peptides: SignalP 3.0.2004In: J Mol Biol, ISSN 0022-2836, Vol. 340, no 4, p. 783-95Article in journal (Refereed)
  • 9.
    Bernsel, Andreas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Viklund, Håkan
    Falk, Jenny
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Prediction of membrane-protein topology from first principles2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 20, p. 7177-7181Article in journal (Refereed)
  • 10. Bernsel, Andreas
    et al.
    Von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Improved membrane protein topology prediction by domain assignments.2005In: Protein Sci, ISSN 0961-8368, Vol. 14, no 7, p. 1723-8Article in journal (Refereed)
  • 11.
    Björkholm, Patrik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ernst, Andreas
    Hacke, Moritz
    Wieland, Felix
    Brügger, Britta
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Identification of novel sphingolipid-binding motifs in mammalian membrane proteinsManuscript (preprint) (Other academic)
    Abstract [en]

    Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif- probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising full proteomes from mammalian organisms. Four selected candidate proteins all tested positive for sphingolipid binding in a photoaffinity assay. The putative sphingolipid-binding motifs are noticeably enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. 

  • 12.
    Björkholm, Patrik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ernst, Andreas M.
    Hacke, Moritz
    Wieland, Felix
    Bruegger, Britta
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Identification of novel sphingolipid-binding motifs in mammalian membrane proteins2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 8, p. 2066-2070Article in journal (Refereed)
    Abstract [en]

    Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.

  • 13.
    Botelho, Salome C.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger R.
    Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues2015In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1848, no 2, p. 615-621Article in journal (Refereed)
    Abstract [en]

    Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.

  • 14.
    Botelho, Salome
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Österberg, Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reichert, Andreas
    Yamano, Koju
    Björkholm, Patrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Endo, Toshiya
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Insertion of model helices into the mitochondrial inner membrane: the rules of the gameManuscript (preprint) (Other academic)
  • 15.
    Botelho, Salomé Calado
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Österberg, Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reichert, Andreas S.
    Yamano, Koji
    Björkholm, Patrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Endo, Toshiya
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    TIM23-mediated insertion of transmembrane alpha-helices into the mitochondrial inner membrane2011In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 30, no 6, p. 1003-1011Article in journal (Refereed)
    Abstract [en]

    While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) alpha-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.

  • 16.
    Calado Botelho, Salomé
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tatsuta, Takashi
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Seoul National University, South Korea.
    Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 7, p. 4792-4798Article in journal (Refereed)
    Abstract [en]

    Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.

  • 17.
    Cassel, Marika
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Seppälä, Susanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Confronting fusion protein-based membrane protein topology mapping with reality: the Escherichia coli ClcA H+/Cl- exchange transporter.2008In: J Mol Biol, ISSN 1089-8638, Vol. 381, no 4, p. 860-6Article in journal (Refereed)
    Abstract [en]

    The topology of bacterial inner membrane proteins is commonly determined using topology reporters such as alkaline phosphatase and green fluorescent protein fused to a series of C-terminally truncated versions of the protein in question. Here, we report a detailed topology mapping of the Escherichia coli inner membrane H(+)/Cl(-) exchange transporter ClcA. Since the 3-D structure of ClcA is known, our results provide a critical test of the reporter fusion approach and offer new insights into the ClcA folding pathway.

  • 18.
    Cymer, Florian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedman, Rickard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ismail, Nurzian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Exploration of the Arrest Peptide Sequence Space Reveals Arrest-enhanced Variants2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 16, p. 10208-10215Article in journal (Refereed)
    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.

  • 19.
    Cymer, Florian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ismail, Nurzian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Weak pulling forces exerted on N-in-orientated transmembrane segments during co-translational insertion into the inner membrane of Escherichia coli2014In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 10, p. 1930-1934Article in journal (Refereed)
    Abstract [en]

    Transmembrane helices (TMHs) in membrane proteins can be orientated with their N-terminus towards the cytoplasm (N-in), or facing the non-cytoplasmic side (N-out). Most membrane proteins are inserted co-translationally into membranes, aided by Sec-type translocons. Since the final orientation of N-in-and N-out-orientated TMHs differs, they could also interact differently with the translocon and the surrounding membrane during insertion. We measured pulling forces exerted on N-in-orientated TMHs during co-translational insertion into the inner membrane of Escherichia coli. Our results demonstrate that Nin-orientated TMHs experience a weaker pulling force but retain the overall biphasic force profile seen previously for Nout-orientated TMHs (Ismail et al., 2012 [1]).

  • 20.
    Cymer, Florian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Cotranslational folding of membrane proteins probed by arrest-peptide-mediated force measurements2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 36, p. 14640-14645Article in journal (Refereed)
    Abstract [en]

    Polytopic membrane proteins are inserted cotranslationally into target membranes by ribosome-translocon complexes. It is, however, unclear when during the insertion process specific interactions between the transmembrane helices start to form. Here, we use a recently developed in vivo technique to measure pulling forces acting on transmembrane helices during their cotranslational insertion into the inner membrane of Escherichia coli to study the earliest steps of tertiary folding of five polytopic membrane proteins. We find that interactions between residues in a C-terminally located transmembrane helix and in more N-terminally located helices can be detected already at the point when the C-terminal helix partitions from the translocon into the membrane. Our findings pinpoint the earliest steps of tertiary structure formation and open up possibilities to study the cotranslational folding of polytopic membrane proteins.

  • 21.
    Cymer, Florian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    White, Stephen H.
    Mechanisms of Integral Membrane Protein Insertion and Folding2015In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 427, no 5, p. 999-1022Article, review/survey (Refereed)
    Abstract [en]

    The biogenesis, folding, and structure of alpha-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons-often referred to as protein-conducting channels-for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. (C) 2014 Elsevier Ltd. All rights reserved.

  • 22.
    Daley, Daniel O
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapp, Mikaela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Granseth, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Melén, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Global topology analysis of the Escherichia coli inner membrane proteome.2005In: Science, ISSN 1095-9203, Vol. 308, no 5726, p. 1321-3Article in journal (Refereed)
  • 23. Daniels, Robert
    et al.
    Mellroth, Peter
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Neiers, Fabrice
    Normark, Staffan
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Henriques-Normark, Birgitta
    Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 5, p. 3300-3309Article in journal (Refereed)
    Abstract [en]

    Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.

  • 24. Ding, Bo
    et al.
    Kull, Björn
    Liu, Zhurong
    Mottagui-Tabar, Salim
    Thonberg, Håkan
    Gu, Harvest F
    Brookes, Anthony J
    Grundemar, Lars
    Karlsson, Christina
    Hamsten, Anders
    Arner, Peter
    Ostenson, Claes-Göran
    Efendic, Suad
    Monné, Magnus
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Per
    Wahlestedt, Claes
    Human neuropeptide Y signal peptide gain-of-function polymorphism is associated with increased body mass index: possible mode of function.2005In: Regul Pept, ISSN 0167-0115, Vol. 127, no 1-3, p. 45-53Article in journal (Refereed)
  • 25. Drew, David
    et al.
    Newstead, Simon
    Sonoda, Yo
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Iwata, So
    GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae.2008In: Nat Protoc, ISSN 1750-2799, Vol. 3, no 5, p. 784-98Article in journal (Refereed)
    Abstract [en]

    It is often difficult to produce eukaryotic membrane proteins in large quantities, which is a major obstacle for analyzing their biochemical and structural features. To date, yeast has been the most successful heterologous overexpression system in producing eukaryotic membrane proteins for high-resolution structural studies. For this reason, we have developed a protocol for rapidly screening and purifying eukaryotic membrane proteins in the yeast Saccharomyces cerevisiae. Using this protocol, in 1 week many genes can be rapidly cloned by homologous recombination into a 2 micro GFP-fusion vector and their overexpression potential determined using whole-cell and in-gel fluorescence. The quality of the overproduced eukaryotic membrane protein-GFP fusions can then be evaluated over several days using confocal microscopy and fluorescence size-exclusion chromatography (FSEC). This protocol also details the purification of targets that pass our quality criteria, and can be scaled up for a large number of eukaryotic membrane proteins in either an academic, structural genomics or commercial environment.

  • 26.
    Elofsson, Arne
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane protein structure: prediction versus reality.2007In: Annu Rev Biochem, ISSN 0066-4154, Vol. 76, p. 125-40Article in journal (Refereed)
  • 27.
    Emanuelsson, Olof
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brunak, Søren
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nielsen, Henrik
    Locating proteins in the cell using TargetP, SignalP and related tools.2007In: Nat Protoc, ISSN 1750-2799, Vol. 2, no 4, p. 953-71Article in journal (Refereed)
  • 28.
    Emanuelsson, Olof
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristóbal, Susana
    In silico prediction of the peroxisomal proteome in fungi, plants and animals.2003In: J Mol Biol, ISSN 0022-2836, Vol. 330, no 2, p. 443-56Article in journal (Refereed)
  • 29.
    Enquist, Karl
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fransson, Mawritz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Boekel, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bengtsson, Inger
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Geiger, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pettersson, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Sofia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane-integration characteristics of two ABC transporters, CFTR and P-glycoprotein2009In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 387, no 5, p. 1153-1164Article in journal (Refereed)
    Abstract [en]

    To what extent do corresponding transmembrane helices in related integral membrane proteins have different membrane-insertion characteristics? Here, we compare, side-by-side, the membrane insertion characteristics of the 12 transmembrane helices in the adenosine triphosphate-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and the cystic fibrosis transmembrane conductance regulator (CFTR). Our results show that 10 of the 12 CFTR transmembrane segments can insert independently into the ER membrane. In contrast, only three of the P-gp transmembrane segments are independently stable in the membrane, while the majority depend on the presence of neighboring loops and/or transmembrane segments for efficient insertion. Membrane-insertion characteristics can thus vary widely between related proteins.

  • 30. Ezure, Toru
    et al.
    Nanatani, Kei
    Sato, Yoko
    Suzuki, Satomi
    Aizawa, Keishi
    Souma, Satoshi
    Ito, Masaaki
    Hohsaka, Takahiro
    von Heijine, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Utsumi, Toshihiko
    Abe, Keietsu
    Ando, Eiji
    Uozumi, Nobuyuki
    A Cell-Free Translocation System Using Extracts of Cultured Insect Cells to Yield Functional Membrane Proteins2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, p. e112874-Article in journal (Refereed)
    Abstract [en]

    Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

  • 31. Fagerberg, Linn
    et al.
    Jonasson, Kalle
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Uhlen, Mathias
    Berglund, Lisa
    Prediction of the human membrane proteome2010In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 10, no 6, p. 1141-1149Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are key molecules in the cell, and are important targets for pharmaceutical drugs. Few three-dimensional structures of membrane proteins have been obtained, which makes computational prediction of membrane proteins crucial for studies of these key molecules. Here, seven membrane protein topology prediction methods based on different underlying algorithms, such as hidden Markov models, neural networks and support vector machines, have been used for analysis of the protein sequences from the 21 416 annotated genes in the human genome. The number of genes coding for a protein with predicted cc-helical transmembrane region(s) ranged from 5508 to 7651, depending on the method used. Based on a majority decision method, we estimate 5539 human genes to code for membrane proteins, corresponding to approximately 26% of the human protein-coding genes. The largest fraction of these proteins has only one predicted transmembrane region, but there are also many proteins with seven predicted transmembrane regions, including the G-protein coupled receptors. A visualization tool displaying the topologies suggested by the eight prediction methods, for all predicted membrane proteins, is available on the public Human Protein Atlas portal (www.proteinatlas.org).

  • 32.
    Farias-Rico, Jose Arcadio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ruud Selin, Frida
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Myronidi, Ioanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Fruehauf, Marie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 40, p. e9280-E9287Article in journal (Refereed)
    Abstract [en]

    During the last five decades, studies of protein folding in dilute buffer solutions have produced a rich picture of this complex process. In the cell, however, proteins can start to fold while still attached to the ribosome (cotranslational folding) and it is not yet clear how the ribosome affects the folding of protein domains of different sizes, thermodynamic stabilities, and net charges. Here, by using arrest peptides as force sensors and on-ribosome pulse proteolysis, we provide a comprehensive picture of how the distance from the peptidyl transferase center in the ribosome at which proteins fold correlates with protein size. Moreover, an analysis of a large collection of mutants of the Escherichia coli ribosomal protein 56 shows that the force exerted on the nascent chain by protein folding varies linearly with the thermodynamic stability of the folded state, and that the ribosome environment disfavors folding of domains of high net-negative charge.

  • 33.
    Farías-Rico, José Arcadio
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Goetz, Sara Kathrin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marino, Jacopo
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mutational analysis of protein folding inside the ribosome exit tunnel2017In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 591, no 1, p. 155-163Article in journal (Refereed)
    Abstract [en]

    Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide (AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel.

  • 34.
    Fluman, Nir
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tobiasson, Victor
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Stable membrane orientations of small dual-topology membrane proteins2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 30, p. 7987-7992Article in journal (Refereed)
    Abstract [en]

    The topologies of alpha-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on antiparallel homo- or heterodimeric small multidrug resistance proteins and examine whether the individual monomers can undergo reversible topological inversion (flip flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane orientation of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final orientations independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo.

  • 35. Freites, J Alfredo
    et al.
    Tobias, Douglas J
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H
    Interface connections of a transmembrane voltage sensor.2005In: Proc Natl Acad Sci U S A, ISSN 0027-8424, Vol. 102, no 42, p. 15059-64Article in journal (Refereed)
  • 36.
    Galian-Barrueco, Carmen
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Björkholm, Patrik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bulleid, Neil
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 20, p. 16399-16409Article in journal (Refereed)
    Abstract [en]

    Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is marginal: less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.

  • 37. Goel, Suchi
    et al.
    Palmkvist, Mia
    Moll, Kirsten
    Joannin, Nicolas
    Lara, Patricia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Akhouri, Reetesh R.
    Moradi, Nasim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Westman, Mattias
    Angeletti, Davide
    Kjellin, Hanna
    Lehtio, Janne
    Blixt, Ola
    Ideström, Lars
    Gahmberg, Carl G.
    Storry, Jill R.
    Hult, Annika K.
    Olsson, Martin L.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wahlgren, Mats
    RIFINs are adhesins implicated in severe Plasmodium falciparum malaria2015In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 21, no 4, p. 314-317Article in journal (Refereed)
    Abstract [en]

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), bind to RBCs-preferentially of blood group A-to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

  • 38.
    Granseth, Erik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Daley, Daniel O
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapp, Mikaela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Melén, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Experimentally constrained topology models for 51,208 bacterial inner membrane proteins.2005In: J Mol Biol, ISSN 0022-2836, Vol. 352, no 3, p. 489-94Article in journal (Refereed)
  • 39.
    Granseth, Erik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Seppälä, Susanna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rapp, Mikaela
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Daley, Daniel O
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane protein structural biology--how far can the bugs take us?2007In: Mol Membr Biol, ISSN 0968-7688, Vol. 24, no 5-6, p. 329-32Article in journal (Other academic)
  • 40.
    Granseth, Erik
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A study of the membrane-water interface region of membrane proteins.2005In: J Mol Biol, ISSN 0022-2836, Vol. 346, no 1, p. 377-85Article in journal (Refereed)
  • 41.
    Hedin, Linnea E.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Öjemalm, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hennerdal, Aron
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Illergård, Kristoffer
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enquist, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kauko, Anni
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cristobal, Susana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerch-Bader, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context2010In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 396, no 1, p. 221-229Article in journal (Refereed)
    Abstract [en]

    In mammalian cells, most integral membrane proteins are initially inserted into the endoplasmic reticulum membrane by the so-called Sec61 translocon. However, recent predictions suggest that many transmembrane helices (TMHs) in multispanning membrane proteins are not sufficiently hydrophobic to be recognized as such by the translocon. In this study, we have screened 16 marginally hydrophobic TMHs from membrane proteins of known three-dimensional structure. Indeed, most of these TMHs do not insert efficiently into the endoplasmic reticulum membrane by themselves. To test if loops or TMHs immediately upstream or downstream of a marginally hydrophobic helix might influence the insertion efficiency, insertion of marginally hydrophobic helices was also studied in the presence of their neighboring loops and helices. The results show that flanking loops and nearest-neighbor TMHs are sufficient to ensure the insertion of many marginally hydrophobic helices. However, for at least two of the marginally hydrophobic helices, the local interactions are not enough, indicating that post-insertional rearrangements are involved in the folding of these proteins.

  • 42.
    Hermansson, Marika
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Inter-helical hydrogen bond formation during membrane protein integration into the ER membrane.2003In: J Mol Biol, ISSN 0022-2836, Vol. 334, no 4, p. 803-9Article in journal (Refereed)
  • 43.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sato, Yoko
    Lerch Bader, Mirjam
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    A quantitative analysis of translocon-mediated insertion of transmembrane alpha-helicesManuscript (Other academic)
  • 44.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bihlmaier, Karl
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lundin, Carolina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Boekel, Jorrit
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersson, Helena
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Recognition of transmembrane helices by the endoplasmic reticulum translocon2005In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 433, no 7024, p. 377-381Article in journal (Refereed)
    Abstract [en]

    Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein - lipid interactions are critical during translocon-mediated membrane insertion.

  • 45.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meindl-Beinker, Nadja M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bernsel, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sato, Yoko
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lerch-Bader, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Molecular code for transmembrane-helix recognition by the Sec61 translocon2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 450, no 7172, p. 1026-1030Article in journal (Refereed)
    Abstract [en]

    Transmembrane alpha-helices in integral membrane proteins are recognized co-translationally and inserted into the membrane of the endoplasmic reticulum by the Sec61 translocon. A full quantitative description of this phenomenon, linking amino acid sequence to membrane insertion efficiency, is still lacking. Here, using in vitro translation of a model protein in the presence of dog pancreas rough microsomes to analyse a large number of systematically designed hydrophobic segments, we present a quantitative analysis of the position- dependent contribution of all 20 amino acids to membrane insertion efficiency, as well as of the effects of transmembrane segment length and flanking amino acids. The emerging picture of translocon- mediated transmembrane helix assembly is simple, with the critical sequence characteristics mirroring the physical properties of the lipid bilayer.

  • 46.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reithinger, Johannes H
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kim, Hyun
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Analysis of transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae2009In: Journal of molecular biology, ISSN 1089-8638, Vol. 386, no 5, p. 1222-8Article in journal (Refereed)
    Abstract [en]

    What sequence features in integral membrane proteins determine which parts of the polypeptide chain will form transmembrane alpha-helices and which parts will be located outside the lipid bilayer? Previous studies on the integration of model transmembrane segments into the mammalian endoplasmic reticulum (ER) have provided a rather detailed quantitative picture of the relation between amino acid sequence and membrane-integration propensity for proteins targeted to the Sec61 translocon. We have now carried out a comparative study of the integration of N out-C in-orientated 19-residue-long polypeptide segments into the ER of the yeast Saccharomyces cerevisiae. We find that the 'threshold hydrophobicity' required for insertion into the ER membrane is very similar in S. cerevisiae and in mammalian cells. Further, when comparing the contributions to the apparent free energy of membrane insertion of the 20 natural amino acids between the S. cerevisiae and the mammalian ER, we find that the two scales are strongly correlated but that the absolute difference between the most hydrophobic and most hydrophilic residues is approximately 2-fold smaller in S. cerevisiae.

  • 47.
    Hessa, Tara
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Membrane insertion of a potassium-channel voltage sensor.2005In: Science, ISSN 1095-9203, Vol. 307, no 5714, p. 1427-Article in journal (Refereed)
  • 48.
    Ismail, Nurzian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedman, Rickard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lindén, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Charge-driven dynamics of nascent-chain movement through the SecYEG translocon2015In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, no 2, p. 145-149Article in journal (Refereed)
    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.

  • 49.
    Ismail, Nurzian
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedman, Rickard
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schiller, Nina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    A biphasic pulling force acts on transmembrane helices during translocon mediated membrane integration2012In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 19, no 10, p. 1018-1022Article in journal (Refereed)
    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.

  • 50. Jaud, Simon
    et al.
    Fernández-Vidal, Mónica
    Nilsson, IngMarie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Meindl-Beinker, Nadja M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hübner, Nadja C.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tobias, Douglas J.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    White, Stephen H.
    Insertion of short transmembrane helices by the Sec61 translocon2009In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, no 28, p. 11588-11593Article in journal (Refereed)
    Abstract [en]

    The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.

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