Skip to main content
SpringerLink
Log in

Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy

  • Protocol
  • First Online:
Rhodopsin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1271))

Abstract

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state 2H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover 2H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state 2H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition we review optimized conditions for trapping the rhodopsin photointermediates; and we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lagerström MC, Schiöth HB (2008) Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov 7:339–357

    Article  PubMed  Google Scholar 

  2. Teller DC, Okada T, Behnke CA et al (2001) Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry 40:7761–7772

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Okada T, Sugihara M, Bondar A-N et al (2004) The retinal conformation and its environment in rhodopsin in light of a new 2.2 Å crystal structure. J Mol Biol 342:571–583

    Article  CAS  PubMed  Google Scholar 

  4. Li J, Edwards PC, Burghammer M et al (2004) Structure of bovine rhodopsin in a trigonal crystal form. J Mol Biol 343:1409–1438

    Article  CAS  PubMed  Google Scholar 

  5. Choe H-W, Kim YJ, Park JH et al (2011) Crystal structure of metarhodopsin II. Nature 471:651–655

    Google Scholar 

  6. Standfuss J, Edwards PC, D’Antona A et al (2011) The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471:656–660

    Google Scholar 

  7. Deupi X, Edwards P, Singhal A et al (2012) Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc Natl Acad Sci U S A 109:119–124

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Struts AV, Salgado GFJ, Martínez-Mayorga K et al (2011) Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsin activation. Nat Struct Mol Biol 18:392–394

    Article  CAS  PubMed  Google Scholar 

  9. Struts AV, Salgado GFJ, Tanaka K et al (2007) Structural analysis and dynamics of retinal chromophore in dark and Meta I states of rhodopsin from 2H NMR of aligned membranes. J Mol Biol 372:50–66

    Article  CAS  PubMed  Google Scholar 

  10. Struts AV, Salgado GFJ, Brown MF (2011) Solid-state 2H NMR relaxation illuminates functional dynamics of retinal cofactor in membrane activation of rhodopsin. Proc Natl Acad Sci U S A 108:8263–8268

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Xu, X., Struts, A.V., and Brown, M.F. (2014) Generalized model-free analysis of nuclear spin relaxation experiments. eMagRes 3:275–286

    Google Scholar 

  12. Papermaster DS, Dreyer WJ (1974) Rhodopsin content in the outer segment membranes of bovine and frog retinal rods. Biochemistry 13:2438–2444

    Article  CAS  PubMed  Google Scholar 

  13. Tanaka K, Struts AV, Krane S et al (2007) Synthesis of CD3-labeled 11-cis-retinals and application to solid-state deuterium NMR spectroscopy of rhodopsin. Bull Chem Soc Jpn 80:2177–2184

    Google Scholar 

  14. Sperling W, Rafferty CN (1969) Relationship between absorption spectrum and molecular conformations of 11-cis-retinal. Nature 224:591–594

    Article  CAS  Google Scholar 

  15. Garwin GG, Saari JC (2000) High-performance liquid chromatography analysis of visual cycle retinoids. Methods Enzymol 316:313–324

    Article  CAS  PubMed  Google Scholar 

  16. Raubach RA, Franklin LK, Dratz EA (1974) A rapid method for the purification of rod outer segment disk membranes. Vision Res 14:335–337

    Article  CAS  PubMed  Google Scholar 

  17. Hong K, Knudsen PJ, Hubbell WL (1982) Purification of rhodopsin on hydroxyapatite columns, detergent exchange, and recombination with phospholipids. Methods Enzymol 81:144–150

    Article  CAS  PubMed  Google Scholar 

  18. Botelho AV, Gibson NJ, Thurmond RL et al (2002) Conformational energetics of rhodopsin modulated by nonlamellar forming lipids. Biochemistry 41:6354–6368

    Google Scholar 

  19. Brown MF (1997) Influence of non-lamellar forming lipids on rhodopsin. Curr Top Membr 44:285–356

    Article  CAS  Google Scholar 

  20. Brown MF, Chan SI (1996) Bilayer membranes: deuterium and carbon-13 NMR. In: Grant DM, Harris RK (eds) Encyclopedia of nuclear magnetic resonance. Wiley, New York, NY, pp 871–885

    Google Scholar 

  21. Brown MF (1996) Membrane structure and dynamics studied with NMR spectroscopy. In: Merz K Jr, Roux B (eds) Biological membranes. A molecular perspective from computation and experiment. Birkhäuser, Basel, pp 175–252

    Google Scholar 

  22. Gröbner G, Taylor A, Williamson PTF et al (1997) Macroscopic orientation of natural and model membranes for structural studies. Anal Biochem 254:132–138

    Article  PubMed  Google Scholar 

  23. Clark NA, Rothschild KJ, Luippold DA et al (1980) Surface-induced lamellar orientation of multilayer membrane arrays. Biophys J 31:65–96

    Google Scholar 

  24. Nevzorov AA, Moltke S, Heyn MP et al (1999) Solid-state NMR line shapes of uniaxially oriented immobile systems. J Am Chem Soc 121:7636–7643

    Article  CAS  Google Scholar 

  25. Martínez-Mayorga K, Pitman MC, Grossfield A et al (2006) Retinal counterion switch mechanism in vision evaluated by molecular simulations. J Am Chem Soc 128:16502–16503

    Article  PubMed  Google Scholar 

  26. Brown MF (1982) Theory of spin-lattice relaxation in lipid bilayers and biological membranes. 2H and 14N quadrupolar relaxation. J Chem Phys 77:1576–1599

    Article  CAS  Google Scholar 

  27. Trouard TP, Alam TM, Brown MF (1994) Angular dependence of deuterium spin-lattice relaxation rates of macroscopically oriented dilaurylphosphatidylcholine in the liquid-crystalline state. J Chem Phys 101:5229–5261

    Article  CAS  Google Scholar 

  28. Nevzorov AA, Brown MF (1997) Dynamics of lipid bilayers from comparative analysis of 2H and 13C nuclear magnetic resonance relaxation data as a function of frequency and temperature. J Chem Phys 107:10288–10310

    Article  CAS  Google Scholar 

  29. Nevzorov AA, Trouard TP, Brown MF (1998) Lipid bilayer dynamics from simultaneous analysis of orientation and frequency dependence of deuterium spin-lattice and quadrupolar order relaxation. Phys Rev E 58:2259–2281

    Article  CAS  Google Scholar 

  30. Brown MF, Salgado GFJ, Struts AV (2010) Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy. Biochim Biophys Acta 1798:177–193

    Article  CAS  PubMed  Google Scholar 

  31. Mertz B, Struts AV, Feller SE et al (2012) Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation. Biochim Biophys Acta 1818:241–251

    Article  CAS  PubMed  Google Scholar 

  32. Spooner PJR, Sharples JM, Verhoeven MA et al (2002) Relative orientation between the β-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes. Biochemistry 41:7549–7555

    Google Scholar 

  33. Verdegem PJE, Bovee-Geurts PHM, de Grip WJ et al (1999) Retinylidene ligand structure in bovine rhodopsin, metarhodopsin-I, and 10-methylrhodopsin from internuclear distance measurements using 13C-labeling and 1-D rotational resonance MAS NMR. Biochemistry 38:11316–11324

    Article  CAS  PubMed  Google Scholar 

  34. Salgado GFJ, Struts AV, Tanaka K et al (2006) Solid-state 2H NMR structure of retinal in metarhodopsin I. J Am Chem Soc 128:11067–11071

    Article  CAS  PubMed  Google Scholar 

  35. Brown MF (2012) Curvature forces in membrane lipid-protein interactions. Biochemistry 51:9782–9795

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85721, USA

    Andrey V. Struts Ph.D., Udeep Chawla M.S., B.S., Suchithranga M. D. C. Perera B.S. & Michael F. Brown Ph.D., A.B

  2. Laboratory of Biomolecular NMR, St. Petersburg State University, St. Petersburg, 199034, Russia

    Andrey V. Struts Ph.D.

  3. Department of Physics, University of Arizona, Tucson, AZ, 85721, USA

    Michael F. Brown Ph.D., A.B

Authors
  1. Andrey V. Struts Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Udeep Chawla M.S., B.S.
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Suchithranga M. D. C. Perera B.S.
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael F. Brown Ph.D., A.B
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael F. Brown Ph.D., A.B .

Editor information

Editors and Affiliations

  1. Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA

    Beata Jastrzebska

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Struts, A.V., Chawla, U., Perera, S.M.D.C., Brown, M.F. (2015). Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy. In: Jastrzebska, B. (eds) Rhodopsin. Methods in Molecular Biology, vol 1271. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2330-4_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2330-4_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2329-8

  • Online ISBN: 978-1-4939-2330-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation