Retinal Proteins (Rhodopsins) Vision, Bioenergetics, Phototaxis. Bacteriorhodopsin s Photocycle. Bacteriorhodopsin s Photocycle

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1 Molecular chanisms of Photoactivation and Spectral Tuning in Retinal Proteins Emad Tajkhorshid Theoretical and Computational Biophysics Group Beckman Institute University of Illinois at Urbana-Champaign Computational Chemistry GRID Conference, 2003 Retinal Proteins (Rhodopsins) mbrane proteins Seven transmembrane helices Retinal chromophore bound to a lysine via a Schiff base All-trans, protonated retinal Schiff base Polyene, charged, A strongly delocalized electronic structure Vision, Bioenergetics, Phototaxis Retinal chromophore Bacteriorhodopsin s Photocycle The photoreceptor of the eye, rhodopsin Activation of cell signaling pathways photoisomerization 400nm 500nm 600nm distinct absorption spectra color vision bacteriorhodopsin in purple membrane hn proton pump V Bacteriorhodopsin s Photocycle Bacteriorhodopsin s Photocycle 1

2 Bacteriorhodopsin s Photocycle Bacteriorhodopsin s Photocycle Bacteriorhodopsin s Photocycle Photocycle of The photocycle is initiated by photoisomerization of the retinal chromophore, and effectively pumps a proton in every cycle (ms) ms D85-C C-D96 C-E204 D85-C C-D96 C-E204 D85-C 568 3ps C-D96 K 603 C-E204 5ms 1µs 500 fs C-D96 D85-C C-E204 C-D96 D85-C C-E L 543 5ms C-D96 40µs D85-C C-E204 M 410 Questions What determines the maximal absorption of retinal in different protein environments? (spectral tuning in bacterial rhodopsins) What are the dynamics of photoisomerization and the mechanism of light energy storage? (excited state QM/ MD) ow does the isomerization is coupled to other molecular events in the protein? (protein activation mechanism in and Rh) Computational thodology Molecular dynamics simulations Modified force field parameters Combined quantum mechanical / molecular mechanical (QM/) calculations Retinal and several key residues are described by ab initio QM (F/CASSCF). Protein environment is treated by a molecular mechanics force field, (AMBER94). 2

3 QM/ Calculations Color Vision Lys216-RET amiltonian of the QM/ system ˆ 2 = pi i 2 g V 1 a ap a p rap QM V i A ia qˆ q p k Z A r i a p 1 r > j ij A> B Zaq p kap r ap Z A Z r AB B QM cone cells QM Asp85 Asp212 dummy atom QM Visual receptors of the rhodopsin family are classified based on their color sensitivity Color Vision Color is sensed by red, green, and blue rhodopsin visual receptors. Spectral Tuning in Bacterial Rhodopsins Sensory Rhodopsin II () phototaxis Bacteriorhodopsin () proton pump 400nm 500nm absorption spectrum 600nm Their chromophores are the same! 11-cis protonated retinal Schiff base Large blue shift of absorption maximum in (70 nm) A prominent sub-band ow does the protein tune the absorption maximum? 500nm 600nm Structures of and Very similar protein structures; Same chromophore: all trans protonated retinal Schiff base X-ray structures: : Luecke et al., Belrhali et al. : Luecke et al., Royant et al. Very Similar Binding Sites Similar structure Aromatic residues. ydrogen-bond network. (counter-ion asparatates, internal water molecules) T204A/V108M/G130S of produces only 20 nm (30%) spectral shift. Mutation of the entire binding site can account for only 50% of the spectral shift! What is the main determinant of spectral tuning? 3

4 retinal- K205/ QM/ Calculations helix G D201 D212 Refinement of X-ray structures by F (retinal, 2Asp, 3 2 ) Excitation energy calculations by CASSCF for retinal retinal- K205/ helix G Excitation Energies Calculated spectra DE( - ) Spectral shift DE( - ) : 6.1 (exp. 7.2) kcal/mol DE( - ): 1.7 (exp. 4.0) kcal/mol A sub-band in is due to the second excited state ( ). X-ray structures D201 D nm 600nm QM/-optimized ayashi, Tajkhorshid, Schulten, JPC -B (2001) Contributions from Residues chanism of Spectral Tuning Strong electronic reorganization upon excitation T142 isolated T204 in gives a blue-shift T142 in gives a red-shift Consistent with mutagenesis experiments chanism of Spectral Tuning Strong electronic reorganization upon excitation C Asp (Glu) isolated in protein Excited states and the ground state receive different stabilization from a counterion inside the protein Structural Determinant of the Spectral Shift Distance between the Schiff base and the counterion is shorter in. 16 C g (Asp201: ) : 4.5 A 16 C g (Asp212: ) : 5.2 A Decomposed electronic reorganization energies retinal- K205/ D201 D212 Same residue!!!! helix G This is why mutagenesis experiments cannot restore the maximal absorption. ayashi, Tajkhorshid, Schulten, JPC -B (2001) 4

5 Photoisomerization of Retinal ne of the fastest reactions in nature Femtosecond resolution spectroscopy measurements energy Ab Initio Excited State QM/ MD Simulation QM <500 fs all-trans 13-cis Ultrafast rate ( fs) igh bond selectivity in protein: 100% 13-cis photoproduct In solution: 9-cis (2%), 11-cis (14%), 13-cis (1%) Two low-lying states ( and ) in An analogue of retinal (three double bonds, 20 atoms) with CASSCF(6,6)/DZV AMBER94 force field for the protein 11 trajectories starting from initial configurations generated by classical MD ayashi, Tajkhorshid, Schulten, Biophys. J. (2003) Dynamics of Curve Crossing A typical trajectory in Ensemble of Trajectories Emission and absorption energies cis trans Gas phase dynamics Fast isomerization A single crossing event energy all-trans 13-cis In situ dynamics cis trans Relatively slow isomerization Multiple crossing events Larger energy differences and smaller nonadiabatic couplings at crossing points ayashi, Tajkhorshid, Schulten, Biophys. J. (2003) ayashi, Tajkhorshid, Schulten, Biophys. J. (2003) Isomerization events Bond selective Unidirectional isomerization Product formation: Cis product formation is dominant at the first crossing point. Dynamic Spectral Modulations Time evolution of emission spectrum along a non-isomerizing trajectory emission energy Ye et al. (pump -probe) Stimulated emission at 800 nm 200 fs oscillation Rocking around the isomerizing bond Protein Activation Process in Bacteriorhodopsin ps Gate delay time ( fs) Kobayashi et al. (pump -probe at 610 nm) Spectrogram of fast oscillations on a decay curve Low freq. shift of C 13 =C 14 str Time dependent coupling between C-C str. (in-plane 14 bending) and 14 -P ayashi, Tajkhorshid, Schulten, Biophys. J. (2003) 5

6 ydrogen Bond etwork (B) B Rearrangement and Proton Transfer Ab initio QM/ calculation, ayashi and hmine, JPCB 104, (2000) proton transfer energy (kcal/mol) neutral zwitterionic B rearrangement can induce the primary proton transfer. B rearrangement induces the proton transfer. Isomerization! r 1 (ang.) Modeling of Early Intermediates Step 2: Isomerization (MD) Step 3: Geometry refinement (QM/) Early Intermediates of ground state (1C3W; Lueckeet al.) K LT intermediate state (1QK; Edman et al.) C13=C14 cis K BR C 13 =C 14 -trans C 13 =C 14 -cis Step 1: Equilibration Step 4: Spectroscopic in BR (MD) properties (QM/) Two distinct states! K and KL D85 T89 3 ps Significant structural changes in the binding pocket. Discrepancies between the X-ray and FTIR studies. ayashi, Tajkhorshid, Schulten, Biophys. J. (2002) QM/ Intermediate structures QM/ Intermediate structures BR 3 ps K 80 ps KL BR 3 ps K 80 ps KL X-ray X-ray Luecke 1999 Edman 2000 Luecke 1999 Lanyi 2002 Edman 2000 ayashi, Tajkhorshid, Schulten, Biophys. J. (2002) ayashi, Tajkhorshid, Schulten, Biophys. J. (2002) 6

7 Protein Activation Process in Rhodopsin Dihedral potential and Charges Protein: pdb file, 1ZX Lipids: PPC, Palmitoyl tails of Cys322 and Cys323; Water: 6500 water molecules Total: ~40,000 atoms AMD2, CARMm27, PME 1ns equilibration, 10ns relaxation after isomerization Rotational barriers and ground and isomerization dihedral potentials (inset) of the protonated retinal Schiff base QM/ derived partial atomic charges of the ground and excited states Saam, Tajkhorshid, ayashi, Schulten, Biophys. J. (2002) Retinal Isomerization Retinal Isomerization Dihedral energy of retinal Rearrangement of the -bond network and salt bridges in the retinal binding pocket verall backbone twist Ring-chain Dihedral angle of C11=C12 Twist Propagation Saam, Tajkhorshid, ayashi, Schulten, Biophys. J. (2002) Decoupling of Trp265 and the b -ionone ring Different Isomerization Dynamics in Rh and SMD Rotation of elix VI Rh Torque applied to rotate helix VI before (red) and 5ns after (blue) isomerization Saam, Tajkhorshid, ayashi, Schulten, Biophys. J. (2002) 7

8 chanism of Energy Storage Major conformational Changes Movement of helices 10ns after isomerization Cytoplasmic changes Internal (bonded) and interaction energy of retinal with its surrounding eeds longer simulations! Saam, Tajkhorshid, ayashi, Schulten, Biophys. J. (2002) Acknowledgment Shigehiko ayashi (UIUC) Jan Saam (UIUC) Klaus Schulten (UIUC) Ehud Landau (UTMB) Javier avarro (UTMB) $$ FSP, SF, I 8