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Experimentally based topology models for E. coli inner membrane proteins
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2004 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 4, 937-945 p.Article in journal (Refereed) Published
Abstract [en]

Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

Place, publisher, year, edition, pages
2004. Vol. 13, no 4, 937-945 p.
Keyword [en]
membrane proteins, topology, prediction, bioinformatics, fusion protein, PhoA, green fluorescent, protein, FP
Identifiers
URN: urn:nbn:se:su:diva-24327DOI: 10.1110/ps.03553804OAI: oai:DiVA.org:su-24327DiVA: diva2:197241
Note
Part of urn:nbn:se:su:diva-6875Available from: 2007-05-24 Created: 2007-05-15 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Topology Prediction of Membrane Proteins: Why, How and When?
Open this publication in new window or tab >>Topology Prediction of Membrane Proteins: Why, How and When?
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are of broad interest since they constitute a large fraction of the proteome in all organisms, up to 20-30%. They play a crucial role in many cellular processes mediating information flow and molecular transport across otherwise nearly impermeable membranes. Traditional three-dimensional structural analyses of membrane proteins are difficult to perform, which makes studies of other structural aspects important. The topology of an α-helical membrane protein is a two-dimensional description of how the protein is embedded in the membrane and gives valuable information on both structure and function.

This thesis is focused on predicting the topology of α-helical membrane proteins and on assessing and improving the prediction accuracy. Reliability scores have been derived for a number of prediction methods, and have been integrated into the widely used TMHMM predictor. The reliability score makes it possible to estimate the trustworthiness of a prediction.

Mapping the full topology of a membrane protein experimentally is time-consuming and cannot be done on a genome-wide scale. However, determination of the location of one part of a membrane protein relative to the membrane is feasible. We have analyzed the impact of incorporating such experimental information a priori into TMHMM predictions and show that the accuracy increases significantly. We further show that the C-terminal location of a membrane protein (inside or outside) is the optimal information to use as a constraint in the predictions.

By combining experimental techniques for determining the C-terminal location of membrane proteins with topology predictions, we have produced reliable topology models for the majority of all membrane proteins in the model organisms E. coli and S. cerevisiae. The results were further expanded to ~15,000 homologous proteins in 38 fully sequenced eukaryotic genomes. This large set of reliable topology models should be useful, in particular as the structural data for eukaryotic membrane proteins is very limited.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2007. 61 p.
Keyword
membrane protein, topology prediction, bioinformatics
National Category
Theoretical Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-6875 (URN)91-7155-397-5 (ISBN)
Public defence
2007-06-15, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2007-05-24 Created: 2007-05-15Bibliographically approved
2. GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli
Open this publication in new window or tab >>GFP as a tool to monitor membrane protein topology and overexpression in Escherichia coli
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2005. 65 p.
Keyword
GFP, membrane protein, topology, overexpression, high-throughput
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-734 (URN)91-7155-160-3 (ISBN)
Public defence
2005-12-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2005-11-09 Created: 2005-11-09 Last updated: 2012-02-06Bibliographically approved
3. The Ins and Outs of Membrane Proteins: Topology Studies of Bacterial Membrane Proteins
Open this publication in new window or tab >>The Ins and Outs of Membrane Proteins: Topology Studies of Bacterial Membrane Proteins
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

α-helical membrane proteins comprise about a quarter of all proteins in a cell and carry out a wide variety of essential cellular functions. This thesis is focused on topology analyses of bacterial membrane proteins. The topology describes the two-dimensional structural arrangement of a protein relative to the membrane.

By combining large-scale experimental and bioinformatics techniques we have produced experimentally constrained topology models for the major part of the Escherichia coli membrane proteome. This represents a substantial increase in available topology information for bacterial membrane proteins.

Many membrane protein structures show signs of internal duplication and approximate two-fold in-plane symmetry. We propose a step-wise pathway to explain how proteins with such internal inverted repeats have evolved. The pathway is based on the ‘positive-inside’ rule and starts with a protein that can adopt two topologies in the membrane, i.e. a “dual” topology protein. The gene encoding the dual topology protein is duplicated and eventually, through re-distribution of positively charge residues, the two resulting homologous proteins become fixed in opposite orientations in the membrane. Finally, the two proteins may fuse into one single polypeptide with an internal inverted repeat structure.

Finally, we re-create the proposed step-wise evolutionary pathway in the laboratory by showing that only a small number of mutations are required in order to transform the homo-dimeric, dual topology protein EmrE into a hetero-dimeric complex composed of two oppositely oriented proteins.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2006. 58 p.
Keyword
membrane protein, topology, dual topology
National Category
Dentistry
Identifiers
urn:nbn:se:su:diva-1330 (URN)91-7155-311-8 (ISBN)
Public defence
2006-12-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2006-10-29 Created: 2006-10-29 Last updated: 2010-08-25Bibliographically approved

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