Publications by authors named "Gunnar von Heijne"

171 Publications

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli.

J Mol Biol 2021 May 12;433(15):167047. Epub 2021 May 12.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Science for Life Laboratory Stockholm University, Box 101, SE-171 21 Solna, Sweden. Electronic address:

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 ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~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.
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http://dx.doi.org/10.1016/j.jmb.2021.167047DOI Listing
May 2021

The ribosome modulates folding inside the ribosomal exit tunnel.

Commun Biol 2021 May 5;4(1):523. Epub 2021 May 5.

Institute of Biological Information Processing IBI-6, Forschungszentrum Jülich (FZJ), Jülich, Germany.

Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.
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http://dx.doi.org/10.1038/s42003-021-02055-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100117PMC
May 2021

Residue-by-residue analysis of cotranslational membrane protein integration in vivo.

Elife 2021 Feb 8;10. Epub 2021 Feb 8.

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

We follow the cotranslational biosynthesis of three multispanning 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.
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http://dx.doi.org/10.7554/eLife.64302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886326PMC
February 2021

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Protein Sci 2020 10 24;29(10):2028-2037. Epub 2020 Aug 24.

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.
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http://dx.doi.org/10.1002/pro.3927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513700PMC
October 2020

Cotranslational folding cooperativity of contiguous domains of α-spectrin.

Proc Natl Acad Sci U S A 2020 06 8;117(25):14119-14126. Epub 2020 Jun 8.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden;

Proteins synthesized in the cell can begin to fold during translation before the entire polypeptide has been produced, which may be particularly relevant to the folding of multidomain proteins. Here, we study the cotranslational folding of adjacent domains from the cytoskeletal protein α-spectrin using force profile analysis (FPA). Specifically, we investigate how the cotranslational folding behavior of the R15 and R16 domains are affected by their neighboring R14 and R16, and R15 and R17 domains, respectively. Our results show that the domains impact each other's folding in distinct ways that may be important for the efficient assembly of α-spectrin, and may reduce its dependence on chaperones. Furthermore, we directly relate the experimentally observed yield of full-length protein in the FPA assay to the force exerted by the folding protein in piconewtons. By combining pulse-chase experiments to measure the rate at which the arrested protein is converted into full-length protein with a Bell model of force-induced rupture, we estimate that the R16 domain exerts a maximal force on the nascent chain of ∼15 pN during cotranslational folding.
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http://dx.doi.org/10.1073/pnas.1909683117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322005PMC
June 2020

Membrane integration and topology of RIFIN and STEVOR proteins of the Plasmodium falciparum parasite.

FEBS J 2020 07 26;287(13):2744-2762. Epub 2019 Dec 26.

Department of Biochemistry and Biophysics, Stockholm University, Sweden.

The malarial parasite Plasmodium exports its own proteins to the cell surfaces of red blood cells (RBCs) during infection. Examples of exported proteins include members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family of proteins from Plasmodium falciparum. The presence of these parasite-derived proteins on surfaces of infected RBCs triggers the adhesion of infected cells to uninfected cells (rosetting) and to the vascular endothelium potentially obstructing blood flow. While there is a fair amount of information on the localization of these proteins on the cell surfaces of RBCs, less is known about how they can be exported to the membrane and the topologies they can adopt during the process. The first step of export is plausibly the cotranslational insertion of proteins into the endoplasmic reticulum (ER) of the parasite, and here, we investigate the insertion of three RIFIN and two STEVOR proteins into the ER membrane. We employ a well-established experimental system that uses N-linked glycosylation of sites within the protein as a measure to assess the extent of membrane insertion and the topology it assumes when inserted into the ER membrane. Our results indicate that for all the proteins tested, transmembranes (TMs) 1 and 3 integrate into the membrane, so that the protein assumes an overall topology of Ncyt-Ccyt. We also show that the segment predicted to be TM2 for each of the proteins likely does not reside in the membrane, but is translocated to the lumen.
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http://dx.doi.org/10.1111/febs.15171DOI Listing
July 2020

The Mgr2 subunit of the TIM23 complex regulates membrane insertion of marginal stop-transfer signals in the mitochondrial inner membrane.

FEBS Lett 2020 03 8;594(6):1081-1087. Epub 2019 Dec 8.

School of Biological Sciences, Seoul National University, South Korea.

The TIM23 complex mediates membrane insertion of presequence-containing mitochondrial proteins via a stop-transfer mechanism. Stop-transfer signals consist of hydrophobic transmembrane segments and flanking charges. Mgr2 functions as a lateral gatekeeper of the TIM23 complex. However, it remains elusive which features of stop-transfer signals are discriminated by Mgr2. To determine the effects of Mgr2 on the TIM23-mediated stop-transfer pathway, we measured membrane insertion of model transmembrane segments of varied hydrophobicity and flanking charges in Mgr2-deletion or -overexpression yeast strains. We found that upon deletion of Mgr2, the threshold hydrophobicity for membrane insertion, as well as the requirement for matrix-facing positive charges, is reduced. These results imply that the Mgr2-mediated gatekeeper function is important for controlling membrane sorting of marginal stop-transfer signals.
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http://dx.doi.org/10.1002/1873-3468.13692DOI Listing
March 2020

Detecting sequence signals in targeting peptides using deep learning.

Life Sci Alliance 2019 10 30;2(5). Epub 2019 Sep 30.

Department of Health Technology, Section for Bioinformatics, Technical University of Denmark, Kongen Lyngby, Denmark

In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state-of-the-art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria, and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, that is, the one following the initial methionine, has a strong influence on the classification. We observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position 2, compared with 20% in other plant proteins. We also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. The importance of this feature for predictions has not been highlighted before.
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http://dx.doi.org/10.26508/lsa.201900429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769257PMC
October 2019

Dynamic membrane topology in an unassembled membrane protein.

Nat Chem Biol 2019 10 9;15(10):945-948. Epub 2019 Sep 9.

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

Helical membrane proteins are typically assumed to attain stable transmembrane topologies immediately upon co-translational membrane insertion. Here we show that unassembled monomers of the small multidrug resistance (SMR) family exist in a dynamic equilibrium where the N-terminal transmembrane helix flips in and out of the membrane, with rates that depend on dimerization and the polypeptide sequence. Thus, membrane topology can display rapid dynamics in vivo and can be regulated by post-translational assembly.
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http://dx.doi.org/10.1038/s41589-019-0356-9DOI Listing
October 2019

Structural and mutational analysis of the ribosome-arresting human XBP1u.

Elife 2019 06 27;8. Epub 2019 Jun 27.

Gene Center, Department of Biochemistry, Center for integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich, Germany.

XBP1u, a central component of the unfolded protein response (UPR), is a mammalian protein containing a functionally critical translational arrest peptide (AP). Here, we present a 3 Å cryo-EM structure of the stalled human XBP1u AP. It forms a unique turn in the ribosomal exit tunnel proximal to the peptidyl transferase center where it causes a subtle distortion, thereby explaining the temporary translational arrest induced by XBP1u. During ribosomal pausing the hydrophobic region 2 (HR2) of XBP1u is recognized by SRP, but fails to efficiently gate the Sec61 translocon. An exhaustive mutagenesis scan of the XBP1u AP revealed that only 8 out of 20 mutagenized positions are optimal; in the remaining 12 positions, we identify 55 different mutations increase the level of translational arrest. Thus, the wildtype XBP1u AP induces only an intermediate level of translational arrest, allowing efficient targeting by SRP without activating the Sec61 channel.
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http://dx.doi.org/10.7554/eLife.46267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624018PMC
June 2019

A Brief History of Protein Sorting Prediction.

Protein J 2019 06;38(3):200-216

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

Ever since the signal hypothesis was proposed in 1971, the exact nature of signal peptides has been a focus point of research. The prediction of signal peptides and protein subcellular location from amino acid sequences has been an important problem in bioinformatics since the dawn of this research field, involving many statistical and machine learning technologies. In this review, we provide a historical account of how position-weight matrices, artificial neural networks, hidden Markov models, support vector machines and, lately, deep learning techniques have been used in the attempts to predict where proteins go. Because the secretory pathway was the first one to be studied both experimentally and through bioinformatics, our main focus is on the historical development of prediction methods for signal peptides that target proteins for secretion; prediction methods to identify targeting signals for other cellular compartments are treated in less detail.
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http://dx.doi.org/10.1007/s10930-019-09838-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589146PMC
June 2019

Silencing of Aberrant Secretory Protein Expression by Disease-Associated Mutations.

J Mol Biol 2019 06 14;431(14):2567-2580. Epub 2019 May 14.

Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Electronic address:

Signal recognition particle (SRP) recognizes signal sequences of secretory proteins and targets them to the endoplasmic reticulum membrane for translocation. Many human diseases are connected with defects in signal sequences. The current dogma states that the molecular basis of the disease-associated mutations in the secretory proteins is connected with defects in their transport. Here, we demonstrate for several secretory proteins with disease-associated mutations that the molecular mechanism is different from the dogma. Positively charged or helix-breaking mutations in the signal sequence hydrophobic core prevent synthesis of the aberrant proteins and lead to degradation of their mRNAs. The degree of mRNA depletion depends on the location and severity of the mutation in the signal sequence and correlates with inhibition of SRP interaction. Thus, SRP protects secretory protein mRNAs from degradation. The data demonstrate that if disease-associated mutations obstruct SRP interaction, they lead to silencing of the mutated protein expression.
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http://dx.doi.org/10.1016/j.jmb.2019.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684239PMC
June 2019

SignalP 5.0 improves signal peptide predictions using deep neural networks.

Nat Biotechnol 2019 04 18;37(4):420-423. Epub 2019 Feb 18.

Department of Bio and Health Informatics, Technical University of Denmark, Kgs Lyngby, Denmark.

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.
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http://dx.doi.org/10.1038/s41587-019-0036-zDOI Listing
April 2019

Force-Profile Analysis of the Cotranslational Folding of HemK and Filamin Domains: Comparison of Biochemical and Biophysical Folding Assays.

J Mol Biol 2019 03 7;431(6):1308-1314. Epub 2019 Feb 7.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, Sweden. Electronic address:

We have characterized the cotranslational folding of two small protein domains of different folds-the α-helical N-terminal domain of HemK and the β-rich FLN5 filamin domain-by measuring the force that the folding protein exerts on the nascent chain when located in different parts of the ribosome exit tunnel (force-profile analysis, or FPA), allowing us to compare FPA to three other techniques currently used to study cotranslational folding: real-time FRET, photoinduced electron transfer, and NMR. We find that FPA identifies the same cotranslational folding transitions as do the other methods, and that these techniques therefore reflect the same basic process of cotranslational folding in similar ways.
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http://dx.doi.org/10.1016/j.jmb.2019.01.043DOI Listing
March 2019

Murine astrotactins 1 and 2 have a similar membrane topology and mature via endoproteolytic cleavage catalyzed by a signal peptidase.

J Biol Chem 2019 03 29;294(12):4538-4545. Epub 2019 Jan 29.

From the Department of Biochemistry and Biophysics, Stockholm University 10691 Stockholm, Sweden and

Astrotactin 1 (Astn1) and Astn2 are membrane proteins that function in glial-guided migration, receptor trafficking, and synaptic plasticity in the brain as well as in planar polarity pathways in the skin. Here we used glycosylation mapping and protease protection approaches to map the topologies of mouse Astn1 and Astn2 in rough microsomal membranes and found that Astn2 has a cleaved N-terminal signal peptide, an N-terminal domain located in the lumen of the rough microsomal membranes (topologically equivalent to the extracellular surface in cells), two transmembrane helices, and a large C-terminal lumenal domain. We also found that Astn1 has the same topology as Astn2, but we did not observe any evidence of signal peptide cleavage in Astn1. Both Astn1 and Astn2 mature through endoproteolytic cleavage in the second transmembrane helix; importantly, we identified the endoprotease responsible for the maturation of Astn1 and Astn2 as the endoplasmic reticulum signal peptidase. Differences in the degree of Astn1 and Astn2 maturation possibly contribute to the higher levels of the C-terminal domain of Astn1 detected on neuronal membranes of the central nervous system. These differences may also explain the distinct cellular functions of Astn1 and Astn2, such as in membrane adhesion, receptor trafficking, and planar polarity signaling.
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http://dx.doi.org/10.1074/jbc.RA118.007093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433051PMC
March 2019

Cotranslational Folding of a Pentarepeat β-Helix Protein.

J Mol Biol 2018 12 27;430(24):5196-5206. Epub 2018 Oct 27.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; Science for Life Laboratory Stockholm University, Box 1031, SE-171 21 Solna, Sweden. Electronic address:

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum-a homolog of the fluoroquinolone resistance protein MfpA-using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.
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http://dx.doi.org/10.1016/j.jmb.2018.10.016DOI Listing
December 2018

Transmembrane but not soluble helices fold inside the ribosome tunnel.

Nat Commun 2018 12 7;9(1):5246. Epub 2018 Dec 7.

Estructura de Recerca Interdisciplinar en Biotecnologia i Biomedicina (ERI BioTecMed), Departament de Bioquímica i Biologia Molecular, Universitat de València, E-46100, Burjassot, Spain.

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 α-helices before integration. Co-translational folding of nascent chains into an α-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 α-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.
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http://dx.doi.org/10.1038/s41467-018-07554-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286305PMC
December 2018

The shape of the bacterial ribosome exit tunnel affects cotranslational protein folding.

Elife 2018 11 26;7. Epub 2018 Nov 26.

Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.

The ribosome exit tunnel can accommodate small folded proteins, while larger ones fold outside. It remains unclear, however, to what extent the geometry of the tunnel influences protein folding. Here, using ribosomes with deletions in loops in proteins uL23 and uL24 that protrude into the tunnel, we investigate how tunnel geometry determines where proteins of different sizes fold. We find that a 29-residue zinc-finger domain normally folding close to the uL23 loop folds deeper in the tunnel in uL23 Δloop ribosomes, while two ~ 100 residue proteins normally folding close to the uL24 loop near the tunnel exit port fold at deeper locations in uL24 Δloop ribosomes, in good agreement with results obtained by coarse-grained molecular dynamics simulations. This supports the idea that cotranslational folding commences once a protein domain reaches a location in the exit tunnel where there is sufficient space to house the folded structure.
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http://dx.doi.org/10.7554/eLife.36326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298777PMC
November 2018

Folding pathway of an Ig domain is conserved on and off the ribosome.

Proc Natl Acad Sci U S A 2018 11 9;115(48):E11284-E11293. Epub 2018 Nov 9.

Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892;

Proteins that fold cotranslationally may do so in a restricted configurational space, due to the volume occupied by the ribosome. How does this environment, coupled with the close proximity of the ribosome, affect the folding pathway of a protein? Previous studies have shown that the cotranslational folding process for many proteins, including small, single domains, is directly affected by the ribosome. Here, we investigate the cotranslational folding of an all-β Ig domain, titin I27. Using an arrest peptide-based assay and structural studies by cryo-EM, we show that I27 folds in the mouth of the ribosome exit tunnel. Simulations that use a kinetic model for the force dependence of escape from arrest accurately predict the fraction of folded protein as a function of length. We used these simulations to probe the folding pathway on and off the ribosome. Our simulations-which also reproduce experiments on mutant forms of I27-show that I27 folds, while still sequestered in the mouth of the ribosome exit tunnel, by essentially the same pathway as free I27, with only subtle shifts of critical contacts from the C to the N terminus.
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http://dx.doi.org/10.1073/pnas.1810523115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6275497PMC
November 2018

Forces on Nascent Polypeptides during Membrane Insertion and Translocation via the Sec Translocon.

Biophys J 2018 11 10;115(10):1885-1894. Epub 2018 Oct 10.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California. Electronic address:

During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide experiments and coarse-grained molecular dynamics to measure and elucidate the molecular origins of forces that are exerted on NCs during cotranslational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a readout of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis.
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http://dx.doi.org/10.1016/j.bpj.2018.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303271PMC
November 2018

Effects of protein size, thermodynamic stability, and net charge on cotranslational folding on the ribosome.

Proc Natl Acad Sci U S A 2018 10 17;115(40):E9280-E9287. Epub 2018 Sep 17.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden;

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 ribosomal protein S6 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.
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http://dx.doi.org/10.1073/pnas.1812756115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176590PMC
October 2018

Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins.

J Mol Biol 2018 09 7;430(18 Pt B):3190-3199. Epub 2018 Jul 7.

Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto M5G 0A4, Ontario, Canada; Department of Biochemistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada. Electronic address:

Large clostridial toxins (LCTs) are a family of homologous proteins toxins that are directly responsible for the symptoms associated with a number of clostridial infections that cause disease in humans and in other animals. LCTs damage tissues by delivering a glucosyltransferase domain, which inactivates small GTPases, across the endosomal membrane and into the cytosol of target cells. Elucidating the mechanism of translocation for LCTs has been hampered by difficulties associated with identifying marginally hydrophobic segments that insert into the bounding membrane to form the translocation pore. Here, we directly measured the membrane-insertion partitioning propensity for segments spanning the putative pore-forming region using a translocon-mediated insertion assay and synthetic peptides. We identified membrane-inserting segments, as well as a conserved and functionally important negatively charged residue that requires protonation for efficient membrane insertion. We provide a model of the LCT pore, which provides insights into translocation for this enigmatic family of α-helical translocases.
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http://dx.doi.org/10.1016/j.jmb.2018.07.001DOI Listing
September 2018

Protein Evolution and Design.

Annu Rev Biochem 2018 06;87:101-103

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden; email:

This article introduces the Protein Evolution and Design theme of the Annual Review of Biochemistry Volume 87.
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http://dx.doi.org/10.1146/annurev-biochem-062917-012013DOI Listing
June 2018

Membrane protein serendipity.

J Biol Chem 2018 03;293(10):3470-3476

From the Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm and

My scientific career has taken me from chemistry, via theoretical physics and bioinformatics, to molecular biology and even structural biology. Along the way, serendipity led me to work on problems such as the identification of signal peptides that direct protein trafficking, membrane protein biogenesis, and cotranslational protein folding. I've had some great collaborations that came about because of a stray conversation or from following up on an interesting paper. And I've had the good fortune to be asked to sit on the Nobel Committee for Chemistry, where I am constantly reminded of the amazing pace and often intricate history of scientific discovery. Could I have planned this? No way! I just went with the flow ….
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http://dx.doi.org/10.1074/jbc.X118.001958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846136PMC
March 2018

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Elife 2017 07 28;6. Epub 2017 Jul 28.

Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science, Utrecht University, Utrecht, Netherlands.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.
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http://dx.doi.org/10.7554/eLife.26067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577925PMC
July 2017

Stable membrane orientations of small dual-topology membrane proteins.

Proc Natl Acad Sci U S A 2017 07 11;114(30):7987-7992. Epub 2017 Jul 11.

Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden;

The topologies of α-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.
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http://dx.doi.org/10.1073/pnas.1706905114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544329PMC
July 2017

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

Elife 2017 05 30;6. Epub 2017 May 30.

Gene Center, Department of Biochemistry and Center for integrated Protein Science Munich, Ludwig Maximilian University of Munich, Munich, Germany.

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.
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http://dx.doi.org/10.7554/eLife.25642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449182PMC
May 2017

Gene Duplication Leads to Altered Membrane Topology of a Cytochrome P450 Enzyme in Seed Plants.

Mol Biol Evol 2017 08;34(8):2041-2056

Centre National de la Recherche Scientifique, Institute of Plant Molecular Biology, University of Strasbourg, Strasbourg, France.

Evolution of the phenolic metabolism was critical for the transition of plants from water to land. A cytochrome P450, CYP73, with cinnamate 4-hydroxylase (C4H) activity, catalyzes the first plant-specific and rate-limiting step in this pathway. The CYP73 gene is absent from green algae, and first detected in bryophytes. A CYP73 duplication occurred in the ancestor of seed plants and was retained in Taxaceae and most angiosperms. In spite of a clear divergence in primary sequence, both paralogs can fulfill comparable cinnamate hydroxylase roles both in vitro and in vivo. One of them seems dedicated to the biosynthesis of lignin precursors. Its N-terminus forms a single membrane spanning helix and its properties and length are highly constrained. The second is characterized by an elongated and variable N-terminus, reminiscent of ancestral CYP73s. Using as proxies the Brachypodium distachyon proteins, we show that the elongation of the N-terminus does not result in an altered subcellular localization, but in a distinct membrane topology. Insertion in the membrane of endoplasmic reticulum via a double-spanning open hairpin structure allows reorientation to the lumen of the catalytic domain of the protein. In agreement with participation to a different functional unit and supramolecular organization, the protein displays modified heme proximal surface. These data suggest the evolution of divergent C4H enzymes feeding different branches of the phenolic network in seed plants. It shows that specialization required for retention of gene duplicates may result from altered protein topology rather than change in enzyme activity.
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http://dx.doi.org/10.1093/molbev/msx160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5850782PMC
August 2017

Transmembrane helices containing a charged arginine are thermodynamically stable.

Eur Biophys J 2017 Oct 13;46(7):627-637. Epub 2017 Apr 13.

Department of Physiology and Biophysics and the Center for Biomembrane Systems, University of California, Irvine, CA, 92697-4560, USA.

Hydrophobic amino acids are abundant in transmembrane (TM) helices of membrane proteins. Charged residues are sparse, apparently due to the unfavorable energetic cost of partitioning charges into nonpolar phases. Nevertheless, conserved arginine residues within TM helices regulate vital functions, such as ion channel voltage gating and integrin receptor inactivation. The energetic cost of arginine in various positions along hydrophobic helices has been controversial. Potential of mean force (PMF) calculations from atomistic molecular dynamics simulations predict very large energetic penalties, while in vitro experiments with Sec61 translocons indicate much smaller penalties, even for arginine in the center of hydrophobic TM helices. Resolution of this conflict has proved difficult, because the in vitro assay utilizes the complex Sec61 translocon, while the PMF calculations rely on the choice of simulation system and reaction coordinate. Here we present the results of computational and experimental studies that permit direct comparison with the Sec61 translocon results. We find that the Sec61 translocon mediates less efficient membrane insertion of Arg-containing TM helices compared with our computational and experimental bilayer-insertion results. In the simulations, a combination of arginine snorkeling, bilayer deformation, and peptide tilting is sufficient to lower the penalty of Arg insertion to an extent such that a hydrophobic TM helix with a central Arg residue readily inserts into a model membrane. Less favorable insertion by the translocon may be due to the decreased fluidity of the endoplasmic reticulum (ER) membrane compared with pure palmitoyloleoyl-phosphocholine (POPC). Nevertheless, our results provide an explanation for the differences between PMF- and experiment-based penalties for Arg burial.
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http://dx.doi.org/10.1007/s00249-017-1206-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640460PMC
October 2017