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Melissa David Adam Ossin Rutger Mantingh Supervisor: Antoinette Killian Introduction Integral membrane proteins in Escherichia coli cells Located in the inner membrane of cell wall Vital for cellular functions • Difficult to study Due to hydrophobic and amphiphilic nature Less than 1% of high resolution 3D structures known Periplasm Inner membrane Cytoplasm Which alternative method could be used to study integral proteins? Membrane topology prediction Topology model Membrane topology describes which regions of a polypeptide spans the cell membrane Membrane topology can be predicted protein sequence Membranes were thought to have only one topology How did they prove that dual topology proteins may exist? With the use of (K + R) biases as determinant for membrane protein topology (K + R) bias determination Orientation of membrane is determined Loops in cytoplasm has more positive charged residues (‘positive-inside rule’) Effect of single positively charged residue (K+R) bias close to zero change in orientation of protein Considerable (K+R) bias no effect on orientation of protein Dual topology proteins Dual topology membrane proteins Inserts into the membrane in 2 opposite orientation Five candidates for dual-topology: EmrE, SugE, CrcB, YdgC, YnfA features of these proteins Quite small ~ 100 amino acid residues 4 transmembrane helices Only few positively charged lysine and arginine residues Very small (K + R) bias between loops Evolutionary relationship of membrane proteins Singleton Gene pair Protein 1 Protein 2 Protein 1 Protein 2 Gene pair Singleton Protein 1 Protein 2 Protein 1 Protein 2 Hypothesis Dual topology proteins have no or a very small positive amino acid bias. Therefore, adding or subtracting a single positive amino acid will result in topology changes. Methods(1) How to determine topology? Fusion proteins on Cterminus: PhoA PhoA: enzymatically active only in the preiplasm GFP PhoA GFP: florescent only in cytoplasm GFP Methods (2) How to determine biases? Unbiased proteins are incorporated either way (dualtopology) Biased proteins are incorporated in one way Methods (3) Mutations Addition or substitution of/with positive amino acids (K + R) SugE and EmrE EmrE & SugE SugE and EmrE PhoA EmrE & SugE GFP PhoA GFP SugE and EmrE PhoA EmrE & SugE GFP PhoA GFP Control YdgE YdgF CrcB CrcB YnfA and YdgC YnfA YdgC Dual topology proteins: A single gene or a gene pair Singleton Singleton Protein Protein 1 Protein 1 Protein 2 2 Gene Gene pairpair Protein Protein 1 Protein 1 Protein 2 2 225 fully sequenced genomes scanned for pairs and singletons Determination by : SMR protein family -Both singletons and gene pairs -Singletons around (K+R) bias = 0 -Gene pairs bigger (K+R) bias (Leucine + Arginine) bias of “dual topology” proteins SMR protein family Singleton Protein 1 Protein 2 Gene pair Protein 1 Protein 2 (Leucine + Arginine) bias of “dual topology” proteins CrcB protein family YnfA protein family YdgC protein family (Leusine + Arginine) bias of “dual topology” proteins YdgQ and YdgL protein family Not all proteins are dual topology proteins One protein, two orientations in the membrane DUF606 protein family Most proteins 4 or 5 trans membrane helices. Internally duplicated: 9 or 10 trans membrane helices Internally duplicated protein DUF606 10 Trans Membrane helices N-terminus 36% sequence identity C terminus Orientation of internally duplicated proteins 5 Trans membrane helices protein 4 Trans membrane helices protein Internal duplication topology Dual topology membrane proteins by different gene Discussion Dual topology proteins myth or reality ? Discussion Evolutionary path ?