Melissa David Adam Ossin Rutger Mantingh Supervisor: Antoinette Killian

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

2. Introduction Periplasm Inner membrane Cytoplasm 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. PhoA PhoA GFP GFP Methods(1) How to determine topology? • Fusion proteins on C-terminus: • PhoA: enzymatically active only in the preiplasm • GFP: florescent only in cytoplasm

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. PhoA PhoA GFP GFP SugE and EmrE EmrE & SugE

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

16. Control YdgE YdgF

17. CrcB CrcB

18. YnfA and YdgC YdgC YnfA

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

20. 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

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

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

23. (Leusine + Arginine) bias of “dual topology” proteins YdgQ and YdgL protein family Not all proteins are 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 10 Trans Membrane helices N-terminus C terminus 36% sequence identity

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. Discussion • Dual topology proteins myth or reality ?

30. Discussion • Evolutionary path ?