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Rhodopsins

Rhodopsins. Vision: C-terminus is active in transduction BR, SRI and SRII : C-terminus is dispensable ASR (cyanobacterium) C-terminus is required and plays a role in H+ transfer Soluble transducer interacts with C-terminus. SR in Archaea. Outward tilt of helix F

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Rhodopsins

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  1. Rhodopsins • Vision: C-terminus is active in transduction • BR, SRI and SRII : C-terminus is dispensable • ASR (cyanobacterium) C-terminus is required and plays a role in H+ transfer • Soluble transducer interacts with C-terminus

  2. SR in Archaea • Outward tilt of helix F • Crystal structure of the complex of sensory rhodopsin pSR II with transducer Htr II. Two transducer molecules with each two trans-membrane helices (TM1 and 2) are complexed between the the seven-helix bundles of two sensory rhodopsins (helices A to G).

  3. Methods • ASR1-161: full length Anabaena opsin • ASR1-129: truncated sequence • Cloned with His6 tag in E. coli • Site-directed mutants • Cells + all-trans retinal, lysed, protein purified • Laser flash-induced currents in cells – blank • Laser flash-induced absorption

  4. Results: Spectroscopy • Same DA dark-light for full and truncated ASR. • Dark adaptation rate was 1.5 times slower for full vs truncated ASR. • In purified proteins, photocycle was 20% faster for full (t1/2= 52 ms) vs truncated ASR (t1/2= 61 ms). • In cells, full was slower (t1/2 = 205 ms) vs truncated ASR (t1/2= 109 ms).

  5. Results: Charge Movement • Photocurrents in truncated ASR: fast (K), t~40-100 ms (L), t~ ms (two components, M: Schiff base deprotonation) all towards cytoplasm (inwards). • Charge in full ASR: same, but only fast is inwards, all others are outward. • Difference in charge longer component matches formation of M (DA400), faster component fits L formation (rearrangement of residues, retinal and water). • K, L or both are different in full vs truncated ASR.

  6. Results: Charge Movement without His6 tag • In full ASR M formation follows charge movement outwards. • In truncated ASR during formation of M the charge movement goes inward. • Same results with and without His6 tag

  7. Results: Asp75Glu Mutant • In full ASR there is slow charge movement, indicating that Asp75 is not a strong proton acceptor ASR with outward current. • Asp75Glu fast outward current • Asp75Glu reverses charge movement in truncated ASR. • Asp217 as same effect.

  8. Results: Ser86Asp Mutant • Ser86Asp inverts charge movement in full and truncated D75E ASR. • Outward current is suppresed and goes inwards (cytoplastomic).

  9. Summary • C-terminal regulates direction of charge movement during the formation of L and M • Goes from outwards (full) to inwards (truncated) • Proton movement is through a H-bonding network (different from BR, ASR is more hydrophilic). • Strong coupling between L, M and C-terminal suggests that C-terminal may modulate interaction with water soluble transducer. • Chlamydomonas C-terminal is larger than transmembrane helices which may relay the signal to amplication cascade. • C- terminal is coupled to photoactive site.

  10. Proton circulation During the Photocycle of Sensory Rhodopsin II Sasaki, J. and Spudich, J.L. Biophysical Journal, 77, 2145-2152, 1999

  11. H. salinarum • Orange (487 nm)– attractant • Blue (587 to 373 nm)- repellent

  12. H. salinarum

  13. SRI and SRII • SRI moves H+ outwards • HtrI blocks H+ transfer on SRI • SRII charge movements goes inwards, then outwards

  14. Results: pH change with illumination • SRI pH decrease, proton ejection • ~1 proton /SRI • SRI/HtrI no pH change, HtrI binding supresses pumping • SRII pH had small increase and returned to initial value, protons are taken from outside and then released back

  15. Results: pH change with illumination • Both SRI and SRII are present in transformant colony • SRI ~ 20% that of SRII • pH change observed was different using different wavelenghts 590-yellow SRI 490-blue SRII SRI-yellow SRII-blue

  16. Results: Kinetics • Rate of conversion is faster in SRII-HrtII • Proton uptake and release occur in SRII and SRII-HrtII • Pyranine (trace 4) follow H+, matched O formation and decay. 1.2 s 80 ms SRII M O 70 ms 37 ms SRII HtrII M O

  17. Results: pH dependance Slow O • pH higher than 7, M decay slows (M lives longer) and O signal is smaller • pKa is ~ 7.5 • Important residue X- is protonated and deprotonated. (Asp, Glu, Arg) Slow M

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