helices E and F arise later in the bR photocycle
(39, 40), these structural changes cannot be
critical for the mechanism of proton pumping.
This is because these motions are suppressed
in 3D crystals yet the photocycle is similar to that
of bR in the purple membrane (fig. S3), and the
Asp96→Gly96/ Phe171→Cys171/Phe219→Leu219 bR
triple mutant is constitutively open to the CP yet
is able to pump protons (41).
Retinal isomerization reorients the SB proton
into a hydrophobic cavity while breaking its H
bond to Wat402, both of which lower the proton
affinity of the SB (14). An initially twisted retinal
becomes planar within 290 ns, causing Trp182
and Leu93 to be displaced toward the cytoplasm
and allowing a water molecule to order between
Leu93, Thr89, and the SB in the L state. H-bond
interactions from the protonated SB to Wat452
or Thr89 create a pathway for proton transfer to
Asp85 (Fig. 4B) and explain how the SB makes
contact with Asp85 despite having been turned
toward the CP by photoisomerization. A steric
clash between Ce of Lys216 and Wat402 dislodges this water molecule, triggering the collapse of the water-mediated H-bond network on
the EC side of bR. This allows helix C to bend
toward helix G approximately 10 ms after photoactivation and raises the pKa of Asp85 to the
point where it may spontaneously accept a
proton from the SB. Once a proton is transferred, the Asp85-Thr89 H bond is lost (Fig. 5D),
thus breaking the SB connectivity to the EC side
of the protein. Consequently, diffraction data
spanning five orders of magnitude in time reveal how structural changes in bR achieve unidirectional membrane transport half a century
after Jardetzky first proposed the alternating access framework obeyed by all membrane transporters (1).
REFERENCES AND NOTES
1. O. Jardetzky, Nature 211, 969–970 (1966).
2. J. Tenboer et al., Science 346, 1242–1246 (2014).
3. T. R. Barends et al., Science 350, 445–450 (2015).
4. K. Pande et al., Science 352, 725–729 (2016).
5. U. Haupts, J. Tittor, D. Oesterhelt, Annu. Rev. Biophys. Biomol.
Struct. 28, 367–399 (1999).
6. R. Neutze et al., Biochim. Biophys. Acta 1565, 144–167
7. C. Wickstrand, R. Dods, A. Royant, R. Neutze, Biochim.
Biophys. Acta 1850, 536–553 (2015).
8. Y. Matsui et al., J. Mol. Biol. 324, 469–481 (2002).
9. V. I. Borshchevskiy, E. S. Round, A. N. Popov, G. Büldt,
V. I. Gordeliy, J. Mol. Biol. 409, 813–825 (2011).
10. V. Borshchevskiy et al., Acta Crystallogr. D Biol. Crystallogr. 70,
11. R. Neutze, R. Wouts, D. van der Spoel, E. Weckert, J. Hajdu,
Nature 406, 752–757 (2000).
12. K. Hirata et al., Nat. Methods 11, 734–736 (2014).
13. H. Luecke, H. T. Richter, J. K. Lanyi, Science 280, 1934–1937
14. M. Sheves, A. Albeck, N. Friedman, M. Ottolenghi, Proc. Natl.
Acad. Sci. U.S.A. 83, 3262–3266 (1986).
15. C. H. Chang, R. Jonas, R. Govindjee, T. G. Ebrey, Photochem.
Photobiol. 47, 261–265 (1988).
16. S. Hayashi, E. Tajkhorshid, K. Schulten, Biophys. J. 83,
17. A. N. Bondar, S. Fischer, S. Suhai, J. C. Smith, J. Phys. Chem. B
109, 14786–14788 (2005).
18. K. Edman et al., Nature 401, 822–826 (1999).
19. K. Nass et al., J. Synchrotron Rad. 22, 225–238 (2015).
20. B. Schobert, J. Cupp-Vickery, V. Hornak, S. Smith, J. Lanyi,
J. Mol. Biol. 321, 715–726 (2002).
21. H. G. Khorana, Proc. Natl. Acad. Sci. U.S.A. 90, 1166–1171
22. G. A. Jeffrey, An Introduction to Hydrogen Bonding (Oxford
Univ. Press, 1997).
23. T. Kouyama, T. Nishikawa, T. Tokuhisa, H. Okumura, J. Mol.
Biol. 335, 531–546 (2004).
24. A. Royant et al., Nature 406, 645–648 (2000).
25. K. Edman et al., J. Biol. Chem. 279, 2147–2158 (2004).
26. J. K. Lanyi, B. Schobert, J. Mol. Biol. 365, 1379–1392
27. A. Maeda et al., Biochemistry 41, 3803–3809 (2002).
28. A. Maeda, F. L. Tomson, R. B. Gennis, S. P. Balashov,
T. G. Ebrey, Biochemistry 42, 2535–2541 (2003).
29. A. N. Bondar, J. Baudry, S. Suhai, S. Fischer, J. C. Smith,
J. Phys. Chem. B 112, 14729–14741 (2008).
30. A. Maeda et al., Biochemistry 42, 14122–14129 (2003).
31. R. Pomès, B. Roux, Biophys. J. 71, 19–39 (1996).
32. S. Subramaniam, R. Henderson, Nature 406, 653–657
33. H. Kandori et al., Proc. Natl. Acad. Sci. U.S.A. 98, 1571–1576
34. H. Luecke, B. Schobert, H. T. Richter, J. P. Cartailler, J. K. Lanyi,
Science 286, 255–260 (1999).
35. H. Luecke et al., J. Mol. Biol. 300, 1237–1255 (2000).
36. H. J. Sass et al., Nature 406, 649–653 (2000).
37. M. T. Facciotti et al., Biophys. J. 81, 3442–3455 (2001).
38. T. Wang et al., Structure 21, 290–297 (2013).
39. M. H. Koch et al., EMBO J. 10, 521–526 (1991).
40. M. Andersson et al., Structure 17, 1265–1275
41. J. Tittor et al., J. Mol. Biol. 319, 555–565 (2002).
42. Materials and methods are available as supplementary
materials on Science Online.
We thank K. Oshimo for her help in culturing Halobacterium
salinarum and members of the Engineering Team of RIKEN
SPring-8 Center—especially Y. Shimazu, K. Hata, N. Suzuki, and
T. Kin—for technical support. XFEL experiments were conducted
at BL3 of SACLA, with the approval of the Japan Synchrotron
Radiation Research Institute (JASRI) (proposal numbers
2014B8051, 2015A8047, and 2015B8054). Crystals were checked
for diffraction at BL41XU of SPring-8 with the approval of JASRI
(proposal number 2015A1119). This work was supported by the
X-ray Free-Electron Laser Priority Strategy Program (Ministry of
Education, Culture, Sports, Science and Technology of Japan) and
partially by the Strategic Basic Research Program (JST) and RIKEN
Pioneering Project Dynamic Structural Biology. We acknowledge
computational support from the SACLA High Performance
Computing system and the Mini-K supercomputer system. R.N.
acknowledges financial support from the Swedish Research
Council (grants VR 349-2011-6485 and 2015-00560), the Swedish
Foundation for Strategic Research (grant SSF SRL 10-0036),
and the Knut and Alice Wallenberg Foundation (grant KAW
2012.0284). A.R. acknowledges financial support from the
French National Research Agency (grant ANR-11-JSV5-0009).
T.Ki. is supported by Japan Society for the Promotion of Science
KAKENHI grant 15H05476. P.N. acknowledges support from
the European Community’s Seventh Framework Program
(FP7/2007-2013) under grant 290605 (PSI-FELLOW/COFUND).
J.S. acknowledges support from the Swiss National Science
Foundation project grant (SNF 31003A_159558). G.S. acknowledges
support from the Swiss National Science Foundation (grant SNF
310030_153145) and the NCCR-MUST/FAST program. C.S. is
supported by National Research Foundation of Korea (grants
NRF-2015R1A5A1009962 and NRF-2016R1A2B3010980) and the
POSCO Green Science program. A.-N.B. acknowledges support in
1556 23 DECEMBER 2016 • VOL 354 ISSUE 6319 sciencemag.org SCIENCE
Fig. 6. Conformational changes on the CP side of bR. (A and B) Close-up view of the difference Fourier electron density map immediately to the CP
side of the retinal for (A) Dt = 760 ns and (B) Dt = 1.725 ms. All maps are contoured at ±3.5s. Difference Fourier electron density maps from this viewpoint
are shown for all 13 time points in movie S3. (C) Crystallographic structural models deriving from partial-occupancy refinement are superimposed upon
the resting bR structure (purple, partially transparent) for Dt = 16 ns (blue), 760 ns (red), 36.2 ms (orange), and 1.725 ms (yellow).