Biophysical Reviews

, Volume 11, Issue 2, pp 167–181 | Cite as

Photoreaction pathways and photointermediates of retinal-binding photoreceptor proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy

  • Akira NaitoEmail author
  • Yoshiteru Makino
  • Arisu Shigeta
  • Izuru Kawamura


Photoirradiation solid-state NMR spectroscopy is a powerful means to study photoreceptor retinal-binding proteins by the detection of short-lived photointermediates to elucidate the photoreaction cycle and photoactivated structural changes. An in situ photoirradiation solid-state NMR apparatus has been developed for the irradiation of samples with extremely high efficiency to enable observation of photointermediates which are stationary trapped states. Such observation enables elucidation of the photoreaction processes of photoreceptor membrane proteins. Therefore, in situ photoirradiation is particularly useful study the photocycle of retinal-binding proteins such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) because functional photointermediates have relatively longer half-lives than other photointermediates. As a result, several photointermediates have been trapped as stationary state and their detailed structures and photoreaction cycles have been revealed using photoirradiation solid-state NMR spectroscopy at low temperature. Photoreaction intermediates of bacteriorhodopsin, which functions to provide light-driven proton pump activity, were difficult to trap because the half-lives of the photointermediates were shorter than those of sensory rhodopsin. Therefore, these photointermediates are trapped in a freeze-trapped state at a very low temperature and the NMR signals were observed using a combination of photoirradiation and dynamic nuclear polarization (DNP) experiments.


Sensory rhodopsin Bacteriorhodopsin Photoreaction cycle Photointermediate Photoirradiation solid-state NMR 


Funding information

This work was supported by the Grants-in-Aid for Scientific Research in an Innovative Area (JP16H00756 to AN and JP16H00828 to IK), and by the Grants-in-Aid for Scientific Research (C) (JP15K06963 to AN) and Research (B) (JP18H02387 to IK) from the Japan Society for the Promotion of Science (JSPS).

Compliance with ethical standards

Conflict of interest

Akira Naito declares that he has no conflict of interest. Yoshiteru Makino declares that he has no conflict of interest. Arisu Shigeta declares that she has no conflict of interest. Izuru Kawamura declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Ahuja S, Crocker E, Eilers M, Hornak V, Hirshfeld A, Ziliox M, Syrett N, Reeves PJ, Khorana HG, Sheves M, Smith SO (2009) Location of the retinal chromophore in the activated state of rhodopsin. J Biol Chem 284:10190–10201CrossRefGoogle Scholar
  2. Bajaj VS, Hornstein MK, Kreischer KE, Sirigiri JR, Woskov PP, Mak-Jurkauskas ML, Herzfeld J, Temkin RJ, Griffin RG (2007) 250 GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR. J Magn Reson 189:251–279CrossRefGoogle Scholar
  3. Bajaj VS, Mak-Jurkauskas ML, Belenky M, Herzfeld J, Griffin RG (2009) Functional and shunt state of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR. Proc Natl Acad Sci U S A 106:9244–9249CrossRefGoogle Scholar
  4. Becker-Baldus J, Bamann C, Saxena K, Gustmann H, Brown LJ, Brown RCD, Reiter C, Bamberg E, Wachtveitl J, Schwalbe H, Glaubitz C (2015) Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy. Proc Natl Acad Sci U S A 112:9896–9901CrossRefGoogle Scholar
  5. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958CrossRefGoogle Scholar
  6. Chen X, Spudich JL (2002) Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membrane by gene fusion analysis. Biochemistry 41:3891–3896CrossRefGoogle Scholar
  7. Chizov I, Schmies G, Seidel R, Sydor JR, Lüttenberg B, Engelhard M (1998) The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II. Biophys J 75:999–1009CrossRefGoogle Scholar
  8. Concistrè M, Gansmüller A, McLean N, Johannessen OG, Montesinos IM, Bovee-Geurts PHM, Verdegem P, Lugtenburg J, Brown RCD, DeGrip WJ, Levitt MH (2008) Double-quantum 13C nuclear magnetic resonance of bacteriorhodopsin, the first photointermediate in mammalian vision. J Am Chem Soc 130:10490–10491CrossRefGoogle Scholar
  9. Crocker E, Eilers M, Ahuja S, Hornak V, Hirshfeld A, Sheves M, Smith SO (2006) Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin. J Mol Biol 357:163–172CrossRefGoogle Scholar
  10. Duñach M, Marti T, Khorana HG, Rothschild KJ (1990) UV-visible spectroscopy of bacteriorhodopsin mutants: substitution of Arg-82, Asp-85, Tyr-185, and Asp-212 results in abnormal light-dark adaptation. Proc Natl Acad Sci U S A 87:9873–9877CrossRefGoogle Scholar
  11. Ernst OP, Lodowski DT, Elstner M, Hegemann P, Brown LS, Kandori H (2014) Microbial and animal rhodopsins structures, functions and molecular mechanisms. Chem Rev 114:126–163CrossRefGoogle Scholar
  12. Farrar MR, Lakshmi KV, Smith SO, Brown RS, Raap J, Lugtenburg J, Griffin RG, Herzfeld J (1993) Solid state NMR study of [ε-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization. Biophys J 65:310–315CrossRefGoogle Scholar
  13. Feng X, Verdegem PJE, Edén M, Sandström D, Lee YK, Bovee-Ceurts PHM, deGrip WJ, Lugtenburg J, deGroot HJM, Levitt MH (2000) Determination of a molecular torsional angle in the metarhodopsin-I photointermediate of thodopsin by double-quantum solid-state NMR. J Biomol NMR 16:1–8CrossRefGoogle Scholar
  14. Gordeliy VI, Labahn J, Moukhametzianov R, Etremov R, Granzin J, Sohleslnger R, Büldt G, Savopol T, Scheldig AJ, Klare JP, Engelhard M (2002) Molecular basis of transmembrane signaling by sensory rhodopsin II-transducer complex. Nature 419:484–487CrossRefGoogle Scholar
  15. Govorunova EG, Sineshchekov OA, Li H, Spudich JL (2017) Microbial rhodopsins: diversity, mechanisms, and optogenetic applications. Annu Rev Biochem 86:845–872CrossRefGoogle Scholar
  16. He Y, Krebs MP, Fischer WB, Khorana HG, Rothschild KJ (1993) FTIR difference spectroscopy of the bacteriorhodopsin mutant Tyr-185→Phe: detection of a stable O-like species and characterization of its photocycle at low temperature. Biochemistry 32:2282–2290CrossRefGoogle Scholar
  17. Hoff WD, Jung K-H, Spudich JL (1997) Molecular mechanism of photosignaling by archaeal sensory rhodopsins. Annu Rev Biophys Biomol Struct 26:223–258CrossRefGoogle Scholar
  18. Hu JG, Sun BQ, Petkova AT, Griffin RG, Helzfeld J (1997) The predischarge chromophore in bacteriorhodopsin: a 15N solid-state NMR study of the L photointermediate. Biochemistry 36:9316–9322CrossRefGoogle Scholar
  19. Hu JG, Sun BQ, Bizounok M, Hatcher ME, Lansing JC, Raap J, Verdegem PJE, Lugtenburg J, Giffin RG, Herzfeld J (1998) Early and late M intermediates in the bacteriorhodopsin photocycle: a solid-state NMR study. Biochemistry 37:8088–8096CrossRefGoogle Scholar
  20. Imamoto Y, Shichida Y, Hirayama J, Tomioka H, Kamo N, Yoshizawa T (1992) Chromophors configuration of pharaonis phoborhodopsin and its isomerization on photon absorption. Biochemistry 31:2523–2528CrossRefGoogle Scholar
  21. Inoue K, Tsukamoto T, Sudo Y (2014) Molecular and evolutionary aspects of microbial sensory rhodopsins. Biochim Biophys Acta 1837:562–577CrossRefGoogle Scholar
  22. Ishchenko A, Round E, Borshchevskiy V, Grudinin S, Gushchin I, Klare JP, Remeeva A, Polovinkin V, Utrobin P, Balandin T, Engelhard M, Büldt G, Gordeliy V (2017) New insights on signal propagation by sensory rhodopsin II/transducer complex. Sci Rep 7:41811CrossRefGoogle Scholar
  23. Iwasa T, Tokunaga F, Yoshizawa T (1981) Photochemical reaction of 13-cis-bacteriorhodopsin studied by low temperature spectrophotometry. Photochem Photobiol 33:539–545CrossRefGoogle Scholar
  24. Kalisky O, Goldchmidt CR, Ottolength M (1977) On the photocycle and light adaptation of dark-adapted bacteriorhodopsin. Biophys J 19:185–189CrossRefGoogle Scholar
  25. Kamo N, Shimono K, Iwamoto M, Sudo Y (2001) Photochemistry and photoinduced proton-transfer by pharaonis phoborhodopsin. Biochem Mosc 66:1277–1282CrossRefGoogle Scholar
  26. Kawamura I, Kihara N, Ohmine M, Nishimura K, Tuzi S, Saitô H, Naito A (2007) Solid-state NMR studies of two backbone conformations at Tyr185 as a function of retinal configurations in the dark, light, and pressure adapted bacteriorhodopsin. J Am Chem Soc 129:1016–1017CrossRefGoogle Scholar
  27. Kitajima-Ihara T, Furutani Y, Suzuki D, Ihara K, Kandori H, Honma M, Sudo Y (2008) Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium. J Biol Chem 283:23533–23541CrossRefGoogle Scholar
  28. Lakshimi KV, Farrar MR, Raap J, Lugtenburg J, Griffin RG, Herzfeld J (1994) Solid state 13C and 15N NMR investigation of the N intermediate of bacteriorhodopsin. Biochemistry 33:8853–8857CrossRefGoogle Scholar
  29. Lanyi JK (1997) Mechanism of ion transport across membranes. J Biol Chem 272:31209–31212CrossRefGoogle Scholar
  30. Lanyi JK (2000) Molecular mechanism of ion transport in bacteriorhodopsin: insights from crystallographic, spectroscopic, kinetic, and mutational studies. J Phys Chem B 104:11441–11448CrossRefGoogle Scholar
  31. Lanyi JK (2006) Proton transfers in the bacteriorhodopsin photocycle. Biochim Biophys Acta 1757:1012–1018CrossRefGoogle Scholar
  32. Luecke H, Scobert B, Richter H-T, Carteiller J-P, Lany JK (1999) Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol 291:899–911CrossRefGoogle Scholar
  33. Luecke H, Schobert B, Carteiller J-P, Richter H-T, Rosengarth A, Needleman R, Lanyi JK (2000) Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin. J Mol Biol 300:1237–1255CrossRefGoogle Scholar
  34. Makino Y, Kawamura I, Okitsu T, Wada A, Kamo N, Sudo Y, Ueda K, Naito A (2018) Retinal configuration of ppR intermediates revealed by photoirradiation solid-state NMR and DFT. Biophys J 115:72–83CrossRefGoogle Scholar
  35. Mak-Jurkauskas ML, Bajaj VS, Hornstein MK, Belenky M, Griffin RG, Herzfeld J (2008) Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR. Proc Natl Acad Sci U S A 105:883–888CrossRefGoogle Scholar
  36. McDermott AE, Thompson LK, Winkel C, Farrar MR, Pelletier S, Lugtenburg J, Herzfeld J, Griffin RG (1991) Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states. Biochemistry 30:8366–8371CrossRefGoogle Scholar
  37. Morgan JE, Vakkasonglu AS, Lanyi JK Lugtenburg J, Gennis RB, Maeda A (2012) Structural changes upon deprotonation of the proton release group in the bacteriorhodopsin photocycle. Biophys J 103:444–452CrossRefGoogle Scholar
  38. Moukhametzianov R, Klare JP, Efremov R, Baeken C, Göppner A, Labahn J, Englehard M, Buldt G, Gordelly VI (2006) Development of the signal in sensory rhodopsin and its transfer to the cognate transducer. Nature 440:115–119CrossRefGoogle Scholar
  39. Naito A, Kawamura I (2014) Photoactivated structural changes in photoreceptor membrane proteins as revealed by in situ photoirradiation solid-state NMR spectroscopy. In: Separovic F, Naito A (eds) Advances in Biological Solid-State NMR: Proteins and Membrane Active Peptides. Royal Society of Chemistry, London, pp 387–404CrossRefGoogle Scholar
  40. Naito A, Kawamura I, Javkhlantugs N (2015) Recent solid-state NMR studies of membrane-bound peptides and proteins. Annu Rep NMR Spectrosc 86:333–411CrossRefGoogle Scholar
  41. Naito A, Makino Y, Kawamura I (2018a) In-situ photo irradiation solid-state NMR spectroscopy applied to retinal-binding membrane proteins. In: Webb G (ed) Modern Magnetic Resonance, 2nd edn. Springer, Berlin, pp 537–557Google Scholar
  42. Naito A, Makino Y, Tasei Y, Kawamura I (2018b) Photoirradiation and microwave irradiation NMR spectroscopy. In: The Nuclear Magnetic Resonance Society of Japan (ed) Experimental Approaches of NMR Spectroscopy, Springer, Chap. 5, pp 135–170Google Scholar
  43. Nango E, Royant A, Kubo M, Nakane T, Wlckstrand C, Kimura T, Tanaka T, Tono K, Soug C, Tanaka R et al (2016) A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354:1552–1557CrossRefGoogle Scholar
  44. Oshima K, Shigeta A, Makino Y, Kawamura I, Okitsu T, Wada A, Tuzi S, Iwasa T, Naito A (2015) Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy. Photochem Photobiol Sci 14:1694–1702CrossRefGoogle Scholar
  45. Petkova AT, Hatanaka M, Jaroniec CP, Hu JG, Belenky M, Verhoeven M, Lugtenburg J, Griffin RG, Herzfeld J (2002) Tryptophan interactions in bacteriorhodopsin: a heteronuclear solid-state NMR study. Biochemistry 41:2429–2437CrossRefGoogle Scholar
  46. Rath P, Krebs MP, He Y, Khorana HG, Rothschild KJ (1993) Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr185 → Phe: formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct. Biochemistry 32:2272–2281CrossRefGoogle Scholar
  47. Roepe PD, Ahl PL, Herzheld J, Lugtenburg J, Rothschild KJ (1988) Tyrosine protonation changes in bacteriorhodopsin, a Fourier transform infrared study of BR548 and its primary photoproduct. J Biol Chem 263:5110–5117Google Scholar
  48. Roy S, Kikukawa T, Sharma P, Kamo N (2006) All-optical switching in pharaonis phoborhodopsin protein molecules. IEEE Trans Nanobioscience 5:178–187CrossRefGoogle Scholar
  49. Shimono K, Hayashi T, Ikeura Y, Sudo Y, Iwamoto M, Kamo N (2003) Importance of the broad regional interaction for spectral tuning in Natronobacterium pharaonis phoborhodopsin (sensory rhodopsin II). J Biol Chem 278:23882–23889CrossRefGoogle Scholar
  50. Smith SO, deGroot HJM, Gebhard R, Courtin JML, Lugtenburg J, Herzfeld J, Griffin RG (1989) Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR. Biochemistry 28:8897–8904CrossRefGoogle Scholar
  51. Sonar S, Krebs MP, Khoraba HG, Rothschild KJ (1993) Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185 → Phe: evidence for an equilibrium between bR570 and an O-like species. Biochemistry 32:2263–2271CrossRefGoogle Scholar
  52. Spudich JL (1998) Variations on a molecular switch: transport and sensory signalling by archaeal rhodopsins. Mol Microbiol 28:1051–1058CrossRefGoogle Scholar
  53. Spudich JL, Bogomolni RA (1984) Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature 312:509–513CrossRefGoogle Scholar
  54. Spudich JL, Lucke H (2002) Sensory rhodopsin II: functional insights from structure. Curr Opin Struct Biol 12:540–546CrossRefGoogle Scholar
  55. Sudo Y, Furutani Y, Wada A, Ito M, Kamo N, Kandori H (2005) Steric constraint in the primary photoproduct of an archaeal rhodopsin from regiospecific perturbation of C-D stretching vibration of the retinal chromophore. J Am Chem Soc 127:16036–16037CrossRefGoogle Scholar
  56. Sudo Y, Furutani Y, Kandori H, Spudich JL (2006) Functional importance of the interherical hydration bond between Thy204 and Tyr174 of sensory rhodopsin II and its alteration during the signaling process. J Biol Chem 281:34239–34245CrossRefGoogle Scholar
  57. Suzuki D, Irieda H, Honma M, Kawagishi I, Sudo Y (2010) Phototactic and chemotactic signal transduction by transmembrane receptors and transducers in microorganisms. Sensors 10:4010–4039CrossRefGoogle Scholar
  58. Swartz TE, Szundi I, Spudich JL, Bogomolni (2000) New photointermediates in the two photon signaling pathway of sensory rhodopsin I. Biochemistry 39:15101–15109CrossRefGoogle Scholar
  59. Tateishi Y, Abe T, Tamogami J, Nakao Y, Kikukawa T, Kamo N, Unno M (2011) Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis. Biochemistry 50:2135–2143CrossRefGoogle Scholar
  60. Tomonaga Y, Hidaka T, Kawamura I, Nishio T, Ohsawa K, Okitsu T, Wada A, Sudo Y, Kama N, Ramamoorthy A, Naito A (2011) An active photoreceptor intermediate revealed by in situ photoirradiated solid-state NMR spectroscopy. Biophys J 101:L50–L52CrossRefGoogle Scholar
  61. Wegener A-A, Chizhov I, Engelhard M, Steinhoff H-J (2000) Time-resolved detection of transient movement of helix F in spin-labelled pharaonic sensory rhodopsin II. J Mol Biol 301:881–891CrossRefGoogle Scholar
  62. Yomoda H, Makino Y, Tomonaga Y, Hidaka T, Kawamura I, Okitsu T, Wada A, Sudo Y, Naito A (2014) Color-discriminating retinal configuration of sensory rhodopsin I by photo-irradiation solid-state NMR spectroscopy. Angew Chem Int Ed 53:6960–6964CrossRefGoogle Scholar
  63. Yoshida H, Sudo Y, Shimono K, Iwamoto M, Kamo N (2004) Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II). Photohem Photobiol Sci 3:537–542CrossRefGoogle Scholar

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© International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Graduate School of EngineeringYokohama National UniversityYokohamaJapan

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