1. Melissa David Adam Ossin Rutger Mantingh Supervisor: Antoinette Killian

2. Periplasm Cytoplasm Inner membrane 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

3. Which alternative method could be used to study integral proteins? Membrane topology prediction

4. 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

5. How did they prove that dual topology proteins may exist? With the use of (K + R) biases as determinant for membrane protein topology

6. (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

7. 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

8. Evolutionary relationship of membrane proteins

9. 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.

10. Methods(1) How to determine topology? Fusion proteins on C- terminus: PhoA: enzymatically active only in the preiplasm GFP: florescent only in cytoplasm PhoA GFP PhoA GFP

11. Methods (2) How to determine biases? Unbiased proteins are incorporated either way (dual- topology) Biased proteins are incorporated in one way

12. Methods (3) Mutations Addition or substitution of/with positive amino acids (K + R)

13. SugE and EmrE EmrE & SugE

14. SugE and EmrE EmrE & SugE PhoA GFP PhoA GFP

15. SugE and EmrE EmrE & SugE PhoA GFP PhoA GFP

16. Control YdgEYdgF

17. CrcB

18. YnfA and YdgC YnfA YdgC

19. Dual topology proteins: A single gene or a gene pair

20. 225 fully sequenced genomes scanned for pairs and singletons SMR protein family -Both singletons and gene pairs -Singletons around (K+R) bias = 0 -Gene pairs bigger (K+R) bias Determination by :

21. (Leucine + Arginine) bias of “dual topology” proteins SMR protein family

22. CrcB protein family YnfA protein family YdgC protein family (Leucine + Arginine) bias of “dual topology” proteins

23. YdgQ and YdgL protein family Not all proteins are dual topology proteins (Leusine + Arginine) bias of “dual topology” proteins

24. 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

25. Internally duplicated protein DUF606 36% sequence identity 10 Trans Membrane helices N-terminus C terminus

26. Orientation of internally duplicated proteins 5 Trans membrane helices protein 4 Trans membrane helices protein

27. Internal duplication topology

28. Dual topology membrane proteins by different gene

29. Dual topology proteins myth or reality ? Discussion

30. Evolutionary path ? Discussion