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Doktorsavhandling vid Karolinska Institutet
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Nilsson, Johan
Membrane protein topology : Prediction, experimental mapping and genome-wide analysis
Onsdagen den 2 juni 2004, kl. 10.00.
Samuelssonsalen, Scheelelaboratoriet, Karolinska Institutet.
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ISBN: 91-7349-963-3
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Diss: 04:210
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Abstract:
Membrane proteins constitute a large and important class
of proteins. They are responsible for many crucial processes in living cells,
and about 25% of all proteins encoded by a typical genome are predicted to be
membrane proteins. Despite their importance, very few structures have been determined
at atomic resolution. As an alternative, the topology of a membrane protein
may provide valuable information on e.g. the location of ligand binding sites
on receptors and transporters.
In this thesis, bioinformatics and experimental approaches
have been applied to study membrane protein topologies. A method to identify
reliably predicted topologies or partial topologies, using a consensus of five
different prediction algorithms, has been developed. The approach has been used
in combination with limited fusion protein analysis to rapidly produce topology
models of E. coli inner-membrane proteins. In addition, a comprehensive
statistical analysis has been performed on predicted membrane protein topologies
from the genomes of 108 organisms.
The correlation between the number of independent prediction methods that agree
on a topology model, and the reliability of the predicted topology was investigated.
Analysis on a data set of E. coli inner-membrane proteins with experimentally
determined topologies revealed that predicted topologies were very reliable
(>90% correct) when all methods or all but one agreed on the prediction.
When the consensus approach was applied to all putative membrane proteins in
a number of completely sequenced genomes, we found that highly reliable topology
predictions could be made for nearly half of all membrane proteins in most prokaryotic
organisms. The method was subsequently extended to predict also parts of a global
topology with an equally high reliability. With the partial consensus topology
predictions, the average fraction of prokaryotic membrane proteins for which
either a complete or a partial topology of high reliability could be predicted
was increased to approximately 70%.
Fusion protein analysis has been used in combination
with the consensus topology predictions to reduce the amount of experimental
efforts required to establish a topology model. The topologies of 43 E.
coli inner-membrane proteins have been mapped using the combined approach.
Comparative studies of amino acid distributions in predicted
membrane proteins from a total of 108 genomes were performed. The main objective
of the study was to establish if the so called positive-inside rule (the asymmetric
distribution of Lys and Arg residues between polar loops on opposite sides of
a membrane) is valid for all organisms where the genome has been sequenced.
An algorithm for identification of membrane spanning helices was optimised to
yield a minimum number of incorrect predictions. The optimised method was then
applied to all putative membrane proteins in each genome, and the average frequency
bias of each amino acid residue type between polar segments on opposite sides
of the membrane was estimated. The study strongly suggested that the positive-inside
rule is valid in most, if not all, organisms investigated, although it is less
pronounced in eukaryotes than in eubacteria and archaea.
List of papers
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Consensus predictions of membrane protein topology.
Nilsson J, Persson B, von Heijne G
FEBS Lett,
2000;
486(3):
267-9
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Prediction of partial membrane protein topologies using a consensus approach.
Nilsson J, Persson B, Von Heijne G
Protein Sci,
2002;
11(12):
2974-80
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Rapid topology mapping of Escherichia coli inner-membrane proteins by prediction and PhoA/GFP fusion analysis.
Drew D, Sjostrand D, Nilsson J, Urbig T, Chin CN, de Gier JW, von Heijne G
Proc Natl Acad Sci U S A,
2002;
99(5):
2690-5. Epub 2002 Feb 26
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Experimentally based topology models for E. coli inner membrane proteins.
Rapp M, Drew D, Daley DO, Nilsson J, Carvalho T, Melen K, De Gier JW, Von Heijne G
Protein Sci,
2004;
13(4):
937-45
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Comparative analysis of amino acid distributions in integral membrane proteins from 108 genomes.
Nilsson J, Persson B, von Heijne G
Manuscript
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