| 1. |
- Bernsel, Andreas, et al.
(författare)
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Exploring the inner membrane proteome of Escherichia coli : which proteins are eluding detection and why?
- 2009
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Ingår i: Trends in microbiology. - : Elsevier BV. - 1878-4380 .- 0966-842X. ; 17:10, s. 444-9
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Tidskriftsartikel (refereegranskat)abstract
- Proteins embedded in membranes are important for helping the cell adapt to changes in the extracellular milieu and often play key roles in the life cycles of pathogenic microbes. Bioinformatic predictions can provide an estimate of membrane proteins, but experimental approaches of detection are required for a deeper understanding of their functions. To determine the effectiveness of experimental detection approaches, here we collate and discuss data from available proteomic analyses on the inner (or cytoplasmic) membrane of Escherichia coli. We compile a list of proteins that have been experimentally detected and by comparing this to a predicted proteome we identify membrane proteins that have eluded us experimentally. Limitations of current proteomic analyses together with possible solutions are discussed. We also provide a list of proteins for benchmarking the performance of future proteomic studies.
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| 2. |
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| 3. |
- Bernsel, Andreas, et al.
(författare)
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Prediction of membrane-protein topology from first principles
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:20, s. 7177-7181
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 4. |
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| 5. |
- Bernsel, Andreas, 1978-
(författare)
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Sequence-based predictions of membrane-protein topology, homology and insertion
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins comprise around 20-30% of a typical proteome and play crucial roles in a wide variety of biochemical pathways. Apart from their general biological significance, membrane proteins are of particular interest to the pharmaceutical industry, being targets for more than half of all available drugs. This thesis focuses on prediction methods for membrane proteins that ultimately rely on their amino acid sequence only. By identifying soluble protein domains in membrane protein sequences, we were able to constrain and improve prediction of membrane protein topology, i.e. what parts of the sequence span the membrane and what parts are located on the cytoplasmic and extra-cytoplasmic sides. Using predicted topology as input to a profile-profile based alignment protocol, we managed to increase sensitivity to detect distant membrane protein homologs. Finally, experimental measurements of the level of membrane integration of systematically designed transmembrane helices in vitro were used to derive a scale of position-specific contributions to helix insertion efficiency for all 20 naturally occurring amino acids. Notably, position within the helix was found to be an important factor for the contribution to helix insertion efficiency for polar and charged amino acids, reflecting the highly anisotropic environment of the membrane. Using the scale to predict natural transmembrane helices in protein sequences revealed that, whereas helices in single-spanning proteins are typically hydrophobic enough to insert by themselves, a large part of the helices in multi-spanning proteins seem to require stabilizing helix-helix interactions for proper membrane integration. Implementing the scale to predict full transmembrane topologies yielded results comparable to the best statistics-based topology prediction methods.
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| 6. |
- Bernsel, Andreas, et al.
(författare)
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TOPCONS : consensus prediction of membrane protein topology
- 2009
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 37:Suppl. 2, s. W465-W468
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Tidskriftsartikel (refereegranskat)abstract
- TOPCONS (http://topcons.net/) is a web server for consensus prediction of membrane protein topology. The underlying algorithm combines an arbitrary number of topology predictions into one consensus prediction and quantifies the reliability of the prediction based on the level of agreement between the underlying methods, both on the protein level and on the level of individual TM regions. Benchmarking the method shows that overall performance levels match the best available topology prediction methods, and for sequences with high reliability scores, performance is increased by approximately 10 percentage points. The web interface allows for constraining parts of the sequence to a known inside/outside location, and detailed results are displayed both graphically and in text format.
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| 7. |
- Daniels, Robert, et al.
(författare)
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Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:5, s. 3300-3309
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- Hedin, Linnea E., et al.
(författare)
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Membrane Insertion of Marginally Hydrophobic Transmembrane Helices Depends on Sequence Context
- 2010
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Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 396:1, s. 221-229
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
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| 10. |
- Hessa, Tara, et al.
(författare)
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Molecular code for transmembrane-helix recognition by the Sec61 translocon
- 2007
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 450:7172, s. 1026-1030
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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