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Gunnar Von Heijne - One of the best experts on this subject based on the ideXlab platform.
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Topology Membrane-Protein topology
2020Co-Authors: Gunnar Von HeijneAbstract:In the year 2000, it all seemed so simple: Membrane Proteins of the helix-bundle type were built from long, hydrophobic α-helices that were orientated more or less perpendicularly to the Membrane plane But now, 6 years on and 60 high-resolution MembraneProtein structures later, the picture is not so simple. Membrane-embedded helices can be short, long, kinked or interrupted in the middle of the Membrane, they can cross the Membrane at oblique angles, lie flat on the surface of the Membrane, or even span only a part of the Membrane and then turn back, forming so-called re-entrant loops. Therefore, depending on one's view, one can adopt a more or less complicated definition of topology. Even the concept of a transMembrane helix is not entirely clear-cut -exactly how far must a helix reach towards the waterinterface regions on both sides of the Membrane to qualify as transMembrane? As our picture of the underpinnings of MembraneProtein structure has become more complex, we have also come to appreciate that the topology of Membrane Proteins can evolve in interesting ways, and these might not even be the same for all molecules that have identical amino-acid sequences. Individual transMembrane helices in a Protein might vary in their capability to insert efficiently into the Membrane, giving rise to a statistical distribution of topologies with different numbers of transMembrane helices. Dual-topology Proteins insert into the Membrane in two opposite orientations with an approximate 1:1 stoichiometry. Also, homologous Proteins can evolve to insert with opposite orientations, or homologous Proteins can fuse to form structures with two antiparallel Membrane-spanning domains. It has even been shown that transMembrane helices can dynamically reorient across the Membrane in response to drastic changes in lipid composition. However, whether such reorientation phenomena are also part of the normal functioning of Membrane Proteins is less clear. In this review, I summarize the recent developments in our understanding of Membrane-Protein topology and structure. I aim to provide a (reasonably) integrated view of Membrane-Protein biogenesis, which starts from the basic process of helix integration into a Membrane and moves through Membrane-Protein topology to the full three-dimensional (3D) structure. These issues will be presented in an evolutionary context in an attempt to identify the main types of molecular-scale evolutionary event that have helped shape the extent of Membrane proteomes. What the structures say At the time of writing, the Protein Data Bank held more than 100 high-resolution structures of integral Membrane Proteins of the helix-bundle type. The number of known Membrane-Protein structures is growing exponentially and doubles every ~3 years, trailing the statistics for watersoluble Proteins by ~15 years 2 . Yet, despite this encouraging trend, we are still only scratching the surface of the fold space of Membrane Proteins, and each new structure brings new surprises. As the crystallographers' attention has shifted from the rock-solid electron-and proton-conducting MembraneProtein complexes that are involved in photosynthesis and respiration to the more inherently flexible channel and transport Proteins, we have begun to appreciate the large dynamic changes in helix-helix packing interactions, and even in local helical-non-helical structural transitions that can occur in Membrane Proteins. It can no longer be taken for granted that all transMembrane helices are straight, orientated approximately perpendicularly to the Membrane, or that they pack with each other according to simple 'knobs-into-holes' geometries. Fold space The abstract space of all Protein folds. 'Knobs-into-holes' geometry The classic mode of helix-helix packing in which side-chains on one helix fit into spaces between side chains on the opposite helix. Membrane-Protein topology Gunnar von Heijne Abstract | In the world of Membrane Proteins, topology defines an important halfway house between the amino-acid sequence and the fully folded three-dimensional structure. Although the concept of Membrane-Protein topology dates back at least 30 years, recent advances in the field of translocon-mediated Membrane-Protein assembly, proteome-wide studies of Membrane-Protein topology and an exponentially growing number of highresolution Membrane-Protein structures have given us a deeper understanding of how topology is determined and of how it evolves
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control of Membrane Protein topology by a single c terminal residue
Science, 2010Co-Authors: Susanna Seppala, J. S. Slusky, Pilar Llorisgarcera, Mikaela Rapp, Gunnar Von HeijneAbstract:The mechanism by which multispanning helix-bundle Membrane Proteins are inserted into their target Membrane remains unclear. In both prokaryotic and eukaryotic cells, Membrane Proteins are inserted cotranslationally into the lipid bilayer. Positively charged residues flanking the transMembrane helices are important topological determinants, but it is not known whether they act strictly locally, affecting only the nearest transMembrane helices, or can act globally, affecting the topology of the entire Protein. Here we found that the topology of an Escherichia coli inner Membrane Protein with four or five transMembrane helices could be controlled by a single positively charged residue placed in different locations throughout the Protein, including the very C terminus. This observation points to an unanticipated plasticity in Membrane Protein insertion mechanisms.
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Prediction of Membrane-Protein topology from first principles
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Andreas Bernsel, Gunnar Von Heijne, Håkan Viklund, Jenny Falk, Erik Lindahl, Arne ElofssonAbstract:The current best Membrane-Protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of Membrane Proteins. However, because the insertion of transMembrane helices into the Membrane is the outcome of molecular interactions among Protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of Membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of Membrane-Protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.
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Membrane Protein structure prediction versus reality
Annual Review of Biochemistry, 2007Co-Authors: Arne Elofsson, Gunnar Von HeijneAbstract:Since high-resolution structural data are still scarce, different kinds of theoretical structure prediction algorithms are of major importance in Membrane Protein biochemistry. But how well do the current prediction methods perform? Which structural features can be predicted and which cannot? And what can we expect in the next few years?
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asn and asp mediated interactions between transMembrane helices during translocon mediated Membrane Protein assembly
EMBO Reports, 2006Co-Authors: Nadja M Meindlbeinker, Stephen H White, Ingmarie Nilsson, Carolina Lundin, Gunnar Von HeijneAbstract:Inter-helix hydrogen bonding involving asparagine (Asn, N), glutamine (Gln, Q), aspartic acid (Asp, D) or glutamic acid (Glu, E) can drive efficient di- or trimerization of transMembrane helices in detergent micelles and lipid bilayers. Likewise, Asn–Asn and Asp–Asp pairs can promote the formation of helical hairpins during translocon-mediated Membrane Protein assembly in the endoplasmic reticulum. By in vitro translation of model integral Membrane Protein constructs in the presence of rough microsomes, we show that Asn- or Asp-mediated interactions with a neighbouring transMembrane helix can enhance the Membrane insertion efficiency of a marginally hydrophobic transMembrane segment. Our observations suggest that inter-helix hydrogen bonds can form during Sec61 translocon-assisted insertion and thus could be important for Membrane Protein assembly.
Janwillem De Gier - One of the best experts on this subject based on the ideXlab platform.
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consequences of Membrane Protein overexpression in escherichia coli
Molecular & Cellular Proteomics, 2007Co-Authors: Samuel Wagner, Klaas J. Van Wijk, Louise Baars, Jimmy A Ytterberg, Anja Klussmeier, Claudia Wagner, Olof Nord, Perake Nygren, Janwillem De GierAbstract:Overexpression of Membrane Proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different Membrane Proteins (YidC, YedZ, and LepI) fused to green fluorescent Protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble Protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic Membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of Protein complexes in the cytoplasmic Membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three Membrane Proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed Proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer Membrane Proteins. This was consistent with lowered accumulation levels of secreted Proteins in the three Membrane Protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic Membrane Protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic Membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of Membrane Protein overexpression in E. coli.
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optimization of Membrane Protein overexpression and purification using gfp fusions
Nature Methods, 2006Co-Authors: David A Drew, Dirk Jan Slotboom, Edmund R S Kunji, Mirjam Lerch, Janwillem De GierAbstract:Optimizing conditions for the overexpression and purification of Membrane Proteins for functional and structural studies is usually a laborious and time-consuming process. This process can be accelerated using Membrane Protein–GFP fusions 1–3 , which allows direct monitoring and visualization of Membrane Proteins of interest at any stage during overexpression, solubilization and purification (Fig. 1). The exceptionally stable GFP moiety of the fusion Protein can be used to detect Membrane Proteins by observing fluorescence in whole cells during overexpression, with a detection limit as low as 10 µg of GFP per liter of culture, and in solution during solubilization and purification. Notably, the fluorescence of the GFP moiety can also be detected in standard SDS polyacrylamide gels with a detection limit of less than 5 ng of GFP per Protein band (Fig. 2). In-gel fluorescence allows assessment of the integrity of Membrane Protein–GFP fusions and provides a rapid and generic alternative for the notoriously difficult immunoblotting of Membrane Proteins. With whole-cell and in-gel fluorescence the overexpression potential of many Membrane Protein–GFP fusions can be rapidly assessed and yields of promising targets can be improved. In this protocol the Escherichia coli BL21(DE3)-pET system—the most widely used (Membrane) Protein overexpression system—is used as a platform to illustrate the GFP-based method. The methodology described in this protocol can be transferred easily to other systems.
Ueli Suter - One of the best experts on this subject based on the ideXlab platform.
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epithelial Membrane Protein 2 and epithelial Membrane Protein 3 two novel members of the peripheral myelin Protein 22 gene family
Gene, 1996Co-Authors: Verdon Taylor, Ueli SuterAbstract:Peripheral myelin Protein 22 (PMP22) is expressed by Schwann cells in the peripheral nervous system (PNS), and mutations affecting the PMP22 gene are associated with hereditary motor and sensory neuropathies. We have previously defined the PMP22/EMP/MP20 gene family by characterizing the PMP22-related epithelial Membrane Protein-1 (EMP-1). We now report the identification of two additional members of the same family, epithelial Membrane Protein-2 and epithelial Membrane Protein-3 (EMP-2 and EMP-3). Both cDNA-predicted polypeptides share approx. 40% aa identity with PMP22. In human, EMP-2 and EMP-3 mRNA transcripts are found in most tissues with an expression pattern partially overlapping that of PMP22 and EMP-1. EMP-2 is most prominently expressed in the adult ovary, heart, lung and intestine and in fetal lung. The levels of EMP-3 transcripts are highest in peripheral blood leukocytes, ovary, intestine and various embryonic tissues. In contrast to PMP22 and EMP-1, EMP-2 and EMP-3 expression is detectable in the liver. In vitro transcription-translation generates EMP-2 and EMP-3 polypeptides of 18 kDa which is in agreement with their predicted sizes. Since PMP22 has been implicated in the regulation of cell proliferation and apoptosis, it appears likely that these novel members of the PMP22/EMP/MP20 Protein family are also involved in similar regulatory processes in a variety of tissues.
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epithelial Membrane Protein 1 peripheral myelin Protein 22 and lens Membrane Protein 20 define a novel gene family
Journal of Biological Chemistry, 1995Co-Authors: Verdon Taylor, Andrew A Welcher, Amgen Est Program, Ueli SuterAbstract:Peripheral myelin Protein 22 (PMP22) is expressed in many tissues but mainly by Schwann cells as a component of compact myelin of the peripheral nervous system (PNS). Mutations affecting PMP22 are associated with hereditary motor and sensory neuropathies. Although these phenotypes are restricted to the PNS, PMP22 is thought to play a dual role in myelin formation and in cell proliferation. We describe the cloning and characterization of epithelial Membrane Protein-1 (EMP-1), a putative four-transMembrane Protein of 160 amino acids with 40% amino acid identity to PMP22. EMP-1 and PMP22 are co-expressed in most tissues but with differences in relative expression levels. EMP-1 is most prominently found in the gastrointestinal tract, skin, lung, and brain but not in liver. In the corpus gastricum, EMP-1 Protein can be detected in epithelial cells of the gastric pit and isthmus of the gastric gland in a pattern consistent with plasma Membrane association. EMP-1 and PMP22 mRNA levels are inversely regulated in the degenerating rat sciatic nerve after injury and by growth arrest in NIH 3T3 fibroblasts. The discovery of EMP-1 as the second member of a novel gene family led to the identification of the lens-specific Membrane Protein 20 (MP20) as a third but distant relative. The Proteins of this family are likely to serve similar functions possibly related to cell proliferation and differentiation in a variety of cell types.
Samuel Wagner - One of the best experts on this subject based on the ideXlab platform.
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Tuning Escherichia coli for Membrane Protein overexpression
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Samuel Wagner, Dirk Jan Slotboom, Klaas J. Van Wijk, Mirjam Klepsch, Susan Schlegel, Ansgar Appel, Roger Russell Draheim, Michael Tarry, Martin Högbom, Jan Olov PerssonAbstract:A simple generic method for optimizing Membrane Protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to Membrane Protein overexpression. For unknown reasons, overexpression of many Membrane Proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved Membrane Protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for Membrane Protein overexpression and conveniently allows optimizing overexpression of any given Membrane Protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
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consequences of Membrane Protein overexpression in escherichia coli
Molecular & Cellular Proteomics, 2007Co-Authors: Samuel Wagner, Klaas J. Van Wijk, Louise Baars, Jimmy A Ytterberg, Anja Klussmeier, Claudia Wagner, Olof Nord, Perake Nygren, Janwillem De GierAbstract:Overexpression of Membrane Proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different Membrane Proteins (YidC, YedZ, and LepI) fused to green fluorescent Protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble Protein, GST-GFP. Proteomes of total lysates, purified aggregates, and cytoplasmic Membranes were analyzed by one- and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of Protein complexes in the cytoplasmic Membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three Membrane Proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed Proteins, chaperones (DnaK/J and GroEL/S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer Membrane Proteins. This was consistent with lowered accumulation levels of secreted Proteins in the three Membrane Protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic Membrane Protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic Membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a down-regulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of Membrane Protein overexpression in E. coli.
Arne Elofsson - One of the best experts on this subject based on the ideXlab platform.
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Prediction of Membrane-Protein topology from first principles
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Andreas Bernsel, Gunnar Von Heijne, Håkan Viklund, Jenny Falk, Erik Lindahl, Arne ElofssonAbstract:The current best Membrane-Protein topology-prediction methods are typically based on sequence statistics and contain hundreds of parameters that are optimized on known topologies of Membrane Proteins. However, because the insertion of transMembrane helices into the Membrane is the outcome of molecular interactions among Protein, lipids and water, it should be possible to predict topology by methods based directly on physical data, as proposed >20 years ago by Kyte and Doolittle. Here, we present two simple topology-prediction methods using a recently published experimental scale of position-specific amino acid contributions to the free energy of Membrane insertion that perform on a par with the current best statistics-based topology predictors. This result suggests that prediction of Membrane-Protein topology and structure directly from first principles is an attainable goal, given the recently improved understanding of peptide recognition by the translocon.
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Membrane Protein structure prediction versus reality
Annual Review of Biochemistry, 2007Co-Authors: Arne Elofsson, Gunnar Von HeijneAbstract:Since high-resolution structural data are still scarce, different kinds of theoretical structure prediction algorithms are of major importance in Membrane Protein biochemistry. But how well do the current prediction methods perform? Which structural features can be predicted and which cannot? And what can we expect in the next few years?