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Defining the inner membrane proteome of E coli and thereby improving 51,208 topology models of bacterial inner membrane proteins Erik Granseth, Mikaela Rapp, Daniel O. Daley, Karin Melén, David Drew and Gunnar von Heijne The inner membrane proteome of E coli + more Erik Granseth1, Mikaela Rapp2, Daniel O. Daley2, Susanna Seppälä2, Karin Melén1, David Drew2 and Gunnar von Heijne1,2 1Stockholm Bioinformatics Center, AlbaNova, [email protected] 2Department of Biophysics and Biochemistry, Stockholm University Outline Global topology analysis of the Escherichia coli inner membrane proteome Science (2005) 308, 1321-1323 (Experimentally constrained topology models for 51,208 bacterial inner membrane proteins J. Mol. Biol. (2005), 352, 489-494) Dual topology membrane proteins Global topology analysis of the Escherichia coli inner membrane proteome Daniel O. Daley, Mikaela Rapp, Erik Granseth, Karin Melén, David Drew and Gunnar von Heijne Introduction Cytoplasm C Membrane 30Å Periplasm Membrane proteins are found in membranes such as the plasma membrane, mitochondrial membranes, chloroplast membranes, the ER etc. Cytoplasm N A topology model of the membrane protein above N Periplasm C Experimental technique When expressed: only GFP or PhoA will be active and thereby reveal the location of the C-terminus (cytoplasm or periplasm) for a particular membrane protein Periplasm – outside cell S N GFP is only flourescent in the cytoplasm S PhoA is only active in the periplasm N Cytoplasm – inside cell TMHMM was used to create the topology models • TransMembrane Hidden Markov Model • Reliability Score (Melén, J. Mol. Biol. 2003) – A measure of the reliability for a single prediction – Useful for discriminating between good and bad topology predictions • Possibility to constrain predictions Example of constraining TMHMM to a specific location Original prediction Constrained prediction GFP/PhoA activities are used to determine the C-terminal location C in t u Co C 601 proteins for which we can assign the C-terminal Distribution of membrane proteins Most are involved in transport in or out of the cell, but many have unknown function Most membrane proteins have the C-terminal in the cytoplasm They also prefer to have even number of TMH, i.e. N and C in the cytoplasm GFP flourescence provide a good estimate of the amount of fusion protein inserted into the membrane Overexpression potential Toxicity TMHMM performance on 82 known topologies 100 Original TMHMM prediction Fixed Cterm prediction 75 50 We can see an increase in topology prediction performance if we fix the Cterminal 25 0 Correct Cterm Correct Topology When comparing our results of the C-terminal location with these known structures, just one protein is incorrect Conclusions • We have derived high-quality topology models for 601 (almost all) E coli membrane proteins with an error rate of ~1% • We have estimated the overexpression potential and suggest that a large fraction can be produced in sufficient quantities for biochemical and structural work • The final constrained topology models are now deposited in the uniprot database Experimentally constrained topology models for 51,208 bacterial inner membrane proteins • We used the membrane proteins with experimentally determined C-terminal location and searched for homologs in 225 fully sequenced prokaryotic genomes. • We created 51,208 much improved topology models • These cover ~30% of all predicted inner membrane proteins in the 225 genomes Dual topology membrane proteins Mikaela Rapp, Erik Granseth Susanna Seppälä and Gunnar von Heijne The positive inside rule Positive inside rule: Cytoplasm K N R R KKR K Periplasm (K+R) bias = 7-4 = 3 R R R C The majority of (K+R) are situated in the cytoplasm K The (K+R) bias is the difference between (K+R) in the cytoplasm and the periplasm Dual topology Most membrane proteins adopt one unique topology... ...but there is some evidence that dual topology exists Nin Cytoplasm Cin + Periplasm Nout Cout Dual topology means that a membrane protein can insert either way into the membrane. This results in two different topologies for the same sequence. The SMR family Small Multidrug Resistance family YdgE and YdgF have a clear topology SugE and EmrE may have dual topology Cytoplasm C N + N C YdgE YdgF Periplasm • • • • Small 3-4 predicted TMhelices Few K+R residues Very small K+R bias between inside and outside loops YdgF YdgC YnfA CrcB YdgE SugE EmrE (K+R) alterations SMR family = R or K = Charge mutation = TM helix The SMR family EmrE and SugE are highly sensitive and (K+R) alterations lead to big GFP or PhoA differences For the control, YdgE/F, the (K+R) alterations have little effect CrcB, YnfA and YdgC CrcB is also highly sensitive to (K+R) alterations YnfA and YdgC are less sensitive to alterations because they have more K+R than EmrE, SugE and CrcB Dual topology proteins have ~0 (K+R) bias while 2 closely spaced pairs have large +/singleton 2 adjacent copies SMR and CrcB proteins form anti-parallel dimers composed either of two separately expressed and oppositely oriented homologues or of a single dual topology protein YnfA and YdgC do not have 2 adjacent homologs Conclusions • A small number of strategically located mutations are required to redesign the topology of a protein • Some proteins adopt a dual topology, others are duplicated and followed by divergent topology evolution. This results in pairs of oppositely oriented molecules Final conclusions • The work presented here is an important framework for many future studies of membrane proteins • Incorporation of experimental topology information improves the topology models • These papers have been a nice cooperation between experimentalists and bioinformaticians, where both have benefited from each others results Acknowledgements • Gunnar von Heijne • Arne Elofsson • SBC and DBB co-workers