1. Lecture 16: Membrane Proteins

2. Membrane proteins: 1) Overview: Types and Properties 2) Getting into the Membrane 3)What Membrane Proteins Do-- examples 3) Working with Membrane Proteins

3. Membrane Protein Overview -Membrane proteins are 25-35% of the genome. -Often important therapeutic targets: involved in signaling, transport, etc. -Can be anywhere from 25% (neurons) to 75% (mitochondria) by mass of the total membrane. -Under represented structurally

4. Beta-Barrel Alpha Helix Amphipathic Helix Fatty acid Chain GPI-Anchored Peripheral Types of Membrane Proteins

5. Beta-Barrel Proteins

6. Alpha Helical Membrane Proteins -Can classify by number of transmembrane segments (multi vs single pass) and their topology in the membrane. -Different methods are used to predict if a polypeptide chain will cross a membrane.

7. Hydropathy Plots for Alpha Helical Proteins: Classic Method For alpha-helical membrane proteins, you can use hydropathy plots to predict the probability that a segment will be within the membrane. These are generated by measuring, for each amino acid, its partition coefficient between water and a non-interacting, isotropic phase such as ethanol, and calculating from that partition coefficient a transfer free energy.

8. Hydropathy Plots: New Method Von Heijne, Biochem Soc. Trans. 2011

9. Membrane Attachment by a Lipid Anchor -Common for proteins involved in signaling. -A fatty acid chain is attached via a amide or thioester linkage, and anchors the protein in the membrane.

10. Membrane proteins: 1) Overview: Types and Properties 2) Getting into the Membrane 3)What Membrane Proteins Do-- examples 3) Working with Membrane Proteins

11. Getting into the Membrane: Destination Matters!

12. Challenges of co-translational protein targeting Lots of ribosomesSelect the right ribosomeTranslation is fast

13. SRP and its receptor act as molecular matchmakers SR SRP SR ribosome SRP ribosome SRP SR ribosome SRP ribosome 7-12 residue hydrophobic core basic N-terminus Signal peptides

14. The core protein targeting machinery is conserved Eukaryotic (Human) Bacterial (E. coli) Ffh FtsY SR SRP54 SRP68 SRP72 SRP19 SRP9 SRP14 SRPSRP Receptor SRP-Type GTPases

15. Crystal Structure of the Prokaryotic SRP/SR Ataide et al., Science 2011

16. SRP SR Egea et. al.,Nature, 2004 and Focia et al., Science, 2004 SRP and SR are GTPases

17. GTP hydrolysis and the SRP targeting cycle SR SRP SR ribosome SRP SR ribosome GTP GDP

18. Co-translational protein targeting: Structure of RNC-translocon complex Frauenfeld et al., PNAS 2011

19. Structure of the Translocon van den Berg et al., Nature 2004 Top viewSide view

20. Translocon features: the plug and the lateral gate van den Berg et al., Nature 2004

21. Translocon activity! van den Berg et al., Nature 2004

22. Getting into the ER: Single N- terminal TM Segment

23. Getting into the ER: Positive Inside Rule

24. The Problem with Tail Anchored Proteins:

25. Post Translational Targeting: Tail Anchored Proteins Hedge and Keenan, 2011

26. The GET Complex Targets Tail- Anchored Proteins Hedge and Keenan, 2011

27. Post-translational Protein Targeting in Prokaryotes Cross et al., 2009 Very hydrophobic and basic signal peptides go through SRP-dependent pathway. Others go through SecB Folded proteins go through the Tat pathway

28. Model for SecA-mediated Protein Translocation Zimmer et al., Nature 2008 How does SecA use the energy of ATP hydrolysis to push a polypeptide through the translocon? Structural studies suggest that a “two helix finger” with a tyrosine paddle pushes the polypeptide into the translocon. It’s actions are coordinated with that of the peptide binding clamp, which has different conformational states during ATP hydrolysis.

29. Twin Arginine Transporter: Transports Folded Proteins Palmer and Berks Nature Reviews Microbiology 2012 Signal sequence contains twin arginines

30. Chloroplasts and Mitochondria Have Their Own Genome but Most Proteins are Made in the Cytosol and Imported.

31. Trafficking to Mitochondrial Membranes

32. Membrane proteins: 1) Overview: Types and Properties 2) Getting into the Membrane 3)What Membrane Proteins Do-- examples 4) Working with Membrane Proteins

33. Functions of Membrane Proteins: Signaling Transporters Enzymes Anchors

34. Signaling: IRE1 and the Unfolded Protein Response

35. Kawaguchi and NG, Science 30 September 2011

36. Transporters: Aquaporins Gonen et al., Nature 2005

37. Enzymes: FtsH, a Membrane Bound Protease Krzywda et al., Structure 2002 and Bieniossek et al., PNAS 2009

38. Membrane proteins: 1) Overview: Types and Properties 2) Getting into the Membrane 3)What Membrane Proteins Do-- examples 4) Working with Membrane Proteins

39. Working with Membrane Proteins: Pick Detergents Wisely

40. Proteoliposomes

41. Hegde RSHegde RS, Keenan RJ Nature Reviews cell Biology 2011.J Nature Reviews cell Biology 2011. Not Many Membrane Protein Crystal Structures Solved

42. Lipidic Cubic Phase for Membrane Proteins Ehud M. LandauEhud M. Landau and Jürg P. Rosenbusch, PNAS 1996, and Nollert et al., FEBS Letters 2001usch, PNAS 1996, a

43. Electron Crystallography: An Example (Wza) Nesper et al.,JBC 2003 Neg. Stain Diffraction Pattern 2-D crystalsClass averages of single particles

44. Electron Crystallography: Aquaporin Gonen et al., Nature 2005

45. Electron Crystallography: Solved Structures Wisedchaisri et al., Nature 2005