1. Bacteriorhodopsin The Purple Membrane Protein Mike Goodreid CHEM*4550 seminar

2. Outline of Presentation Introduce Bacteriorhodopsin (BR) History of its structural analysis Structural features of the protein Mechanism of action Energy involved in action

3. 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)

4. 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

5. 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

6. 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

7. 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

8. 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

9. Hartmut’s Experiment

10. 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)

11. 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

12. 1990 First structure of BR First structures of BR from side and top/bottom

13. 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

14. Intro to CLP CLP matrix (bicontinuous cubic phase) Involves -high lipid content monoolein (1-monooleoyl-rac-glycerol, C 18:1c9, = MO ) -aqueous pores that penetrate membrane -proteins embedded At high concentrations of lipids, more complex phase behaviour occurs (say goodbye to micelles and bilayers)

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

16. 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)

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

18. 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?)

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

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

21. 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

22. 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.

23. Photocycle A lesson in pushing protons

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

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

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

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

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

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

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

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

32. Basic Biophysics And now for something completely different

33. Thermodynamics of Transport Energy of a photon: E=hc/lambda let lambda = 550 nm E photon =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 Since Ephoton>/\G, we can see that the photon is sufficiently energized to move the proton

34. 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

35. References Lanyi, J.K. (2001) Biochemistry (Moscow) 66, 1477-1482 Brown, L.S. (2001) Biochemistry (Moscow) 66, 1546-1552 Dunn, R., McCoy, J., Simsek, M., Majumdar, A., Chang, S.H., Rajbhandary, U.L., and Khorana, H.G..(1981) Proc Natl Acad Sci USA.78, 6744-6748. Jagannathan, K., Chang, R., and Yethiraj, A. (2002) Biophys J 83, 1902-1916 Peck, R.F., Echavarri-Erasun, C., Johnson, E.A., Ng, W.V., Kennedy S.P., Hood, L., DasSarma, S., and Krebs, M.P. (2001) J Biol Chem. 23, 5739-5744 Landau, E.M.and Rosenbusch, J.P. (1996) Proc. Natl. Acad. Sci. USA 93, 14532-14535 Nollert, P. Qiu, H., Caffrey, M., Rosenbusch, J.P., and Landau, E.M. (2001) FEBS Lett. 504, 179-186