1. Bacteriorhodopsin + H D85-COO HOOC-E204 HOOC-D96 N + H D85-COO HOOC-E204 HOOC-D96 N K216 bR 568 K 603 + H D85-COO HOOC-E204 HOOC-D96 N K216 D85-COOH OOC-E204 HOOC-D96 N K216 L 543 M 410 + H D85-COOH OOC-E204 OOC-D96 N K216 N 550 + H D85-COOH OOC-E204 HOOC-D96 N K216 O 645 3ps  s 40  s 5ms Cytoplasmic side Extracellular side H+H+ H+H+ Photoabsorption -> isomerization -> proton transfer

2. Chromophore-binding Pocket counterions Aromatic amino acids

3. H N + H N + photo-isomerization ~500 fs all-trans 13-cis cytoplasmic extracellular H+H+ Proton accessibility!

4. All structural details in the retinal chromophore are functionally important  -ionone ring Methyl groups conjugated backbone Protonated Schiff base PA = E AH – E A

5. Delocalization of positive charge

6. Low barriers against double bond isomerization Ground state isomerization Isomerization barriers in retinal

7. A twisted chromophore is also experimentally reported. X-ray structures of bR report the twisted form of chromophore The twist is found around the terminal double bonds It may influence pK a of the chromophore 165°168° 178° 177°176°177° A twisted chromophore in bR?

8. Photocycle of bR Photo-induced All intermediates are trapped in low temperature and have been characterized by vibrational and absorption spectroscopy. 3 ps  s 40  s 5 ms

9. Ultrafast spectroscopy 1 fs : 3 x 10 -4 mm 1 ps : 3 x 10 -1 mm

10. Ultrafast spectroscopy of bR

11. Kobayashi et al., Nature, Nov 2001 Femtosecond time resolution Ultrafast spectroscopy of bR 3 ps  s 40  s 5 ms H----I 460 ----J 625

12. Ab initio (First-Principles) dynamics of ethylene in vacuum HOMO 0 fs 50 fs 110 fs LUMO Calculation of the Excited state Dynamics of Photoactive Molecules by ab initio Techniques Todd Martinez, Chemistry, UIUC But what happens in the protein?

13. N O H N H … MMQM N H … H H H dummy atom N O H MM O O QM Lys216-RET Asp85, 212 QM/MM calculations QM MM

14. S0S0 S1S1 K BR C 13 =C 14 -transC 13 =C 14 -cis Coupling of electronic excitation and conformational change in bR 7911 13 15

15. Hydrogen bond network in the retinal binding pocket N K216 H + O H O D85 H O T89 H O H H H O H O O D212 H O Y185 H O Y57 H N H R82 + W402 W401 W406 O H N + H N + 500 fs

16. D85 T89 D212 D85 T89 D212 3 ps Structure of the ground state (1C3W.pdb) Structure of the first intermediate (1QKO.pdb) all-trans 13-cis Water movement after the photoisomerization One water molecule is dislocated, but where is it? C13=C14 cis

17. D85 D212 T89 BRK 77K KL 135K FTIR spec. (77K)X-ray (110K) D85 D212 T89 D85 D212 T89 3ps80ps N-H: 90 o (D212) N-H: 270 o (T89)  E EX (kcal/mol) (e.g., T89-D85: stronger)(W402 is dislocated, D85 is rotated) -4.0 (-1.8)-2.9 (-1.8) Early intermediates of bR’s photocycle

18. Mechanism of Switching N H C O O H O H O C O H O H D85D212 N H C O O H O H O C O H O H D85D212 + - - - - + H O T89 H O N H C O O O C O H O H D85D212 - - + H O T89 N H C O O O C O H O H D85D212 - - + H O T89.. BRK KLL

19. Role of water in proton transfer proton transfer energy (kcal/mol) zwitter -ionic neutral Rearrangement of the hydrogen-bond network can induce the proton transfer. isomerization QM/MM calculation

20. THE PURPLE MEMBRANE

21. The Purple membrane of Halobacterium salinarum

22. Archaeal Membranes Branched (less vulnerable to oxidation) Etheric bridge, not esteric (less sensitive to hydrolysis) Inverted glycerol stereochemistry Higher resistance to harsh conditions of their habitat: pH, heat, high salt and sulfur, …

23. Bacteriorhodopsin trimer Retinal chromophores Internal water molecules Intra-trimer lipids Squalene molecules Inter-trimer lipids Bulk water MODELING OF THE INTEGRAL PURPLE MEMBRANE

24. Extracellular Basic Acidic : PolarLipids Basic : Acidic : Polar : Lipids Cytoplasmic Charge distribution at different faces of the purple membrane

25. Kinetics of the photocycle is dependent on the lipid composition of the membrane PGP and squalene are necessary for the recovery of normal kinetics of the photocycle after detergent treatment of PM. We have 10 molecules of lipid per bR monomer

26. Helix dislocation at late stages of the photocycle Possible involvement of lipid-protein interaction in the photocycle