1. Structure-Function Relationship of Retinal Proteins

2. Retinal proteins or rhodopsins belong to the superfamily of seven- transmembrane helical (7TM) proteins. Seven helices, with N-terminus on the extracellular side and C-terminus on the cytoplasmic side of the membrane (not necessarily G-protein coupled) A B C D E F G Structure of Retinal Proteins GPCRs

3. Retinal Proteins -- Rhodopsins Covalently linked to a lysine Usually protonated Schiff base all-trans and 11-cis isomers Chromophore

4.  The simplest ion pump in biology  The best characterized membrane protein  The simplest photosynthetic center  Technological applications in molecular electronics Bacteriorhodopsin -- bR  The first membrane protein with a known atomic-detail 3D structures

5. Halobacterium Salinarum The Purple Membrane H+H+ h [H + ] ADP ATP Cytoplasmic sideExtracellular side ATP Synthase h bR role in Bioenergetics Light Proton Gradient

6. Cytoplasmic side Extracellular side Schematic proton path in bacteriorhodopsin Transmambrane helices H+H+ H+H+

7. Active Channels Need a ‘Switch’ Mechanism H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ H+H+ h H+H+ H+H+ What is the switch in bR? How does it work?

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. No membrane protein has been studied as extensively as bR Photo-induced 3 ps  s 40  s 5 ms All intermediates have also been characterized by X-ray crystallography!

10. Cytoplasmic side Extracellular side Schematic proton path in bacteriorhodopsin Transmambrane helices

11. cytoplasmic extracellular H+H+ light driven proton pump + 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 BR’s Photocycle

12. + 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 Kuhlbarandt, Nature, 406,569 (2000) Conformational Change of Helices BR’s Photocycle

13.  bR in the purple membrane Modeling of the protein in lipid bilayers  Chromophore Analysis of the structure Calculation of excited state dynamics  Protein Chromophore-protein interaction QM-MM calculations MD simulation of the photocycle Study of bR at three levels

14. Retinal Schiff base Membrane, covalently bound, chromophore Retinoic Acid Nucleus, receptor site, ligand (no photoactivity) Retinoids Retinal

15.        The necessity of quantum mechanical treatment of the chromophore: Conjugated  -electronic system, delocalization The effect of protein matrix on the ligand 79111315 Unconventioanl chemistry QM is expensive – Most of the time, one needs to use models

16. Proton Affinity: PA= E AH -(E A +E H ) Effect of Conjugation on pK a (Gas Phase Proton Affinity)

17. Proton Affinity: PA= E AH -(E A +E H ) Effect of the methyl groups on pK a No more room for additional methyl groups on the backbone

18. h What is the effect of isomerization?

19. Proton Affinity: PA= E AH -(E A +E H ) cc: B2,B3-di-cis isomerct: B2-s-cis, B3-trans isomer tc: B2-strans, B3-cis isomertt: all-trans isomer. Isomerization State and Proton Affinity Isomerization does not have a strong impact on PA!

20. h What is the effect of isomerization?

21. Water Asp 85 Asp 212 Water Counterion: Asp 85 & Asp 212 WATER pK a : ~8.0 13.0 max : ~400 570 Opsin shift Retinal binding pocket in bR

22. Proton Affinity: PA= E AH -(E A +E H ) Effect of the environment on PA

23. In situ isomerization and pK a

24. 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

25. h trans cis Ground and Excited State Potential Energy Surfaces of Retinal

26. Ab Initio QM/MM Excited State MD Simulation QM Quantum mechanical (QM) treatment of the chromophore, and force field (MM) treatment of the embedding protein

27. Proton Affinity: PA= E AH -(E A +E H ) Low barriers against double bond isomerization Ground state isomerization Isomerization Barriers in retinal

28. 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?