Bacteriorhodopsin

1. Bacteriorhodopsin

2. Archea

3. Archea

4. Source of BR • Archaebacteria Halobacteria Salinarium are the source of bacteriorhodopsin • They are halophilic bacteria (found in very salty water e.g. Great Salt Lake)

5. Source of H.salinarum

6. H. Salinarum Electronmicrograph

7. What is the purple membrane? • The purple membrane patches are areas on the membrane where BR is concentrated • BR absorbs light @ 570 nm (visible green light) • Red and Blue light is reflected, giving membrane its purple colour

8. So what does BR do? • BR functions as a proton pump • Long story short: protons are pumped one at a time from the inside of the cell to the outside • Photons react with a bound retinal group causing conformational change in BR

9. Phototaxis

10. Phototaxis

11. Phototaxis

12. Photons for Protons • Bacteriorhodopsin takes energy from photons • This energy is converted and creates a proton gradient by pumping protons outside the cell • Protons are allowed back into the cell by an ATP synthase • In a nutshell: Photons are used to power the cell

13. Photoactive Proteins in H.salinarum • bacteriorhodopsin(Haupts et. al. (1999), Oesterhelt (1998))the photosynthetic pigment that permits Halobacterium to grow with light as only energy source • a light-driven proton pump which converts light energy into a proton gradient. The energy stored in the proton gradient can be used in different ways, e.g. for generation of ATP via ATP synthase • halorhodopsin(Kolbe et. al. (2000), Oesterhelt (1998))a light-driven chloride pump that permits Halobacterium to maintain the high internal salt concentration upon growth

14. Photoactive proteins in H.salinarum • sensory rhodopsin I • involved in phototaxis, mediates the photophilic response to orange and also the photophobic response to UV light • forms a complex with the transducer protein htrI • sensory rhodopsin II • involved in phototaxis, mediates the photophobic response to blue light • forms a complex with the transducer protein htrII

15. Milestones in BR Structural Determination • In order to assess the structure and mechanism of BR, or any membrane protein, we really need to understand its tertiary structure by X-ray crystallography • BUT, membrane proteins don’t crystallize easily

16. Nobel Prize in Chemistry (1988) • Hartmut Michel • First to crystallize BR in 1980 • Contribution to determination of structure of a photosynthetic reaction center earned him a Nobel Prize

17. Hartmut’s Experiment

18. Findings • Could get protein crystallization • Crystals were too small and disordered to determine tertiary structure • Results uncommon because • BR is a very stable protein • BR forms a 2D lattice in vivo and in vitro (later)

19. 1990 • Henderson et al. use cryo-crystallography to study BR • Crystallization occurred • First instances of structural determination • However, some areas of the protein could not be resolved

20. 1990 First structure of BR First structures of BR from side and top/bottom

21. H. Salinarum Bacteriorhodopsin

22. 1996: E.M Landau & J.P. Rosenbusch • Paradigm shift in crystallization of membrane proteins • Use Cubic Lipid Phase Matrix • First complete structural determination of BR

23. Intro to CLP CLP matrix (bicontinuous cubic phase) Involves -high lipid content monoolein (1-monooleoyl-rac-glycerol, C18:1c9, = MO) -aqueous pores that penetrate membrane -proteins embedded At high concentrations of lipids, more complex phase behaviour occurs (say goodbye to micelles and bilayers)

24. Seeding and Feeding • Purple membrane patches (or BR monomers) diffuse into the CLP • Addition of Sorensen salt increases curvature of the CLP’s membranes

25. Seeding and Feeding • Protein separates into planar domains (crystal formation) • Mature crystals co-exist with BR depleted cubic phase • Hydration (dilution of Sorensen salt solution) reverses the crystallization process (crystals dissolve back into CLP matrix)

26. Results Hexagonal crystals from MO bicontinuous lipid phase lead to complete structural determination of BR (3.7 Angstrom resolution)

27. BR gene expression • 786 nt structural gene • 13 AA precursor sequence +248 AA in mature BR +1 AA (D) at C-terminal sequence • No intervening sequences • No prokaryotic promoter (yet?)

28. Brp has role in retinal synthesis from beta-carotene Blh has a similar role(?)

29. Structural Features of BR Cytosol H+ | | V Extracellular matrix

30. Structural Info • 7 TM helices • Forms a homotrimer • Homotrimers aggregate to form the purple membrane • Stability of trimer by: • G113, I117, L48 • Most stability comes from surrounding lipids

31. Retinal Proteins

32. Bacteriorhodopsin Proton Pump

33. Are There Any Highly-conserved Residues? You’d better believe it! L. Brown, 2001: -Upon BLASTing the H. Salinarium BR, found very high homology among all BR from a number of different Halobacterium -Around the K216 schiff base, there is no deviation in AA composition for a good 4.5 Angstroms -This type of analysis shows the entire retinal binding pocket is highly conserved. Therefore, MANY of the AAs in BR are structurally and/or catalytically important. SDM is a useful tool for validating this statement.

34. Photocycle A lesson in pushing protons

35. Photocycle of Bacteriorhodopsin

36. Photocycle of Bacteriorhodopsin

37. All-trans retinal (blue) Carbon 13 (red) 13 | CHO 13 Photocycle of BR begins with absorption of a photon with wavelength of 550 nm. All-trans retinal13-cis retinal

38. 13-cis retinal (blue+cyan) Carbon 13 (red) 13 | CHO 13 Photocycle of BR begins with absorption of a photon with wavelength of 550 nm. All-trans retinal13-cis retinal

39. Photocycle (K) K Cytosol H H+ | | V H cis PRS H Extracellular matrix

40. Photocycle (L) KL Cytosol H -Partial retinal relaxation -Subtle changes in protein conformation H+ | | V H cis PRS H Extracellular matrix

41. Photocycle (M) LM Cytosol H -K216 (schiff base deprotonated) -D85 picks up proton (perhaps via H2O intermediate) -Proton lost from PRS H+ | | V cis H PRS Extracellular matrix

42. Photocycle (N) MN Cytosol -D96 deprotonated -K216 picks up proton H+ | | V H cis H PRS Extracellular matrix

43. Photocycle (O) NO Cytosol H -Retinal reisomerizes back to All-Trans -D96 reprotonated from cytosol H+ | | V H H PRS Extracellular matrix

44. Photocycle (final step) OK Cytosol H -D85 deprotonated -PRS reprotonated -back to square 1 until another proton isomerizes the All-trans retinal H+ | | V H H PRS Extracellular matrix

45. Retinal Binding Pocket

46. Basic Biophysics And now for something completely different

47. Energy of a photon: E=hc/lambda let lambda = 550 nm Ephoton=3.61x10^(-19) J Energy req’d to move H+ /\G=RTln([H+out]/[H+in]) -zF/\psi let: H+out=10,000 H+in, T=295K /\G=3.75E-20(J/H+) - zF/\psi let: Vm=-60mV (an estimate) /\G=(3.75(E-20) – 9.61E(-21)) J/H+ /\G = + 4.7E-20J Thermodynamics of Transport Since Ephoton>/\G, we can see that the photon is sufficiently energized to move the proton

48. The retinal Chromophore

49. What promise does BR hold? Bioengineering: -Scaffold for a light powered Cation pump -Facilitate environmental cleanup of heavy metals -Cheap, easy way of accumulating protons: -Industry -Fuel cell cars

50. Application of Bacteriorhodopsin