Journal of Biomolecular NMR

, Volume 64, Issue 3, pp 223–237 | Cite as

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

  • Shu Y. Liao
  • Myungwoon Lee
  • Tuo Wang
  • Ivan V. Sergeyev
  • Mei Hong


Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.


Membrane proteins Sensitivity enhancement DNP Influenza M2 AMUPol Paramagnetic relaxation enhancement Solid-state NMR 



The authors thank Prof. Robert Griffin, Eric Keeler and Vladmir Michaelis for stimulating discussions. This work is supported by National Institutes of Health Grants GM088204 and GM066976 to M.H.


  1. Akbey U, Franks WT, Linden A, Orwick-Rydmark M, Lange S, Oschkinat H (2013) Dynamic nuclear polarization enhanced NMR in the solid-state. Top Curr Chem 338:181–228CrossRefGoogle Scholar
  2. Andreas LB, Barnes AB, Corzilius B, Chou JJ, Miller EA, Caporini M, Rosay M, Griffin RG (2013) Dynamic nuclear polarization study of inhibitor binding to the M2(18–60) proton transporter from influenza A. Biochemistry 52:2774–2782CrossRefGoogle Scholar
  3. 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 189:251–279ADSCrossRefGoogle Scholar
  4. Bajaj VS, Mak-Jurkauskas ML, Belenky M, Herzfeld J, Griffin RG (2009) Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR. Proc Natl Acad Sci USA 106:9244–9249ADSCrossRefGoogle Scholar
  5. Barnes AB, De Paepe G, van der Wel PCA, Hu KN, Joo CG, Bajaj VS, Mak-Jurkauskas ML, Sirigiri JR, Herzfeld J, Temkin RJ, Griffin RG (2008) High-field dynamic nuclear polarization for solid and solution biological NMR. Appl Magn Reson 34:237–263CrossRefGoogle Scholar
  6. Becerra LR, Gerfen GJ, Temkin RJ, Singel DJ, Griffin RG (1993) Dynamic nuclear-polarization with a cyclotron-resonance maser at 5 T. Phys Rev Lett 71:3561–3564ADSCrossRefGoogle Scholar
  7. 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 USA 112:9896–9901ADSCrossRefGoogle Scholar
  8. Bloembergen N (1957) Proton relaxation times in paramagnetic solutions—comment. J Chem Phys 27:595–596ADSCrossRefGoogle Scholar
  9. Buffy JJ, Hong T, Yamaguchi S, Waring AJ, Lehrer RI, Hong M (2003) Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+. Biophys J 85:2363–2373CrossRefGoogle Scholar
  10. Cady SD, Wang J, Wu Y, DeGrado WF, Hong M (2011a) Specific binding of adamantane drugs and direction of their polar amines in the pore of the influenza M2 transmembrane domain in lipid bilayers and dodecylphosphocholine micelles determined by NMR spectroscopy. J Am Chem Soc 133:4274–4284CrossRefGoogle Scholar
  11. Cady SD, Wang T, Hong M (2011b) Membrane-dependent effects of a cytoplasmic helix on the structure and drug binding of the influenza virus M2 protein. J Am Chem Soc 133:11572–11579CrossRefGoogle Scholar
  12. Can TV, Ni QZ, Griffin RG (2015) Mechanisms of dynamic nuclear polarization in insulating solids. J Magn Reson 253:23–35ADSCrossRefGoogle Scholar
  13. Carver TR, Slichter CP (1956) Experimental verification of the overhauser nuclear polarization effect. Phys Rev 102:975–980ADSCrossRefGoogle Scholar
  14. Cheng CY, Han SI (2013) Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems. Ann Rev Phys Chem 64:507–532ADSCrossRefGoogle Scholar
  15. Chu S, Maltsev S, Emwas AH, Lorigan GA (2010) Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers. J Magn Reson 207:89–94ADSCrossRefGoogle Scholar
  16. Craven BM (1976) Crystal-structure of cholesterol monohydrate. Nature 260:727–729ADSCrossRefGoogle Scholar
  17. Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM (2005) Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis. J Am Chem Soc 127:12291–12305CrossRefGoogle Scholar
  18. Gerfen GJ, Becerra LR, Hall DA, Griffin RG, Temkin RJ, Singel DJ (1995) High-frequency (140 Ghz) dynamic nuclear-polarization—polarization transfer to a solute in frozen aqueous-solution. J Chem Phys 102:9494–9497ADSCrossRefGoogle Scholar
  19. Hall DA, Maus DC, Gerfen GJ, Inati SJ, Becerra LR, Dahlquist FW, Griffin RG (1997) Polarization-enhanced NMR spectroscopy of biomolecules in frozen solution. Science 276:930–932CrossRefGoogle Scholar
  20. Hu KN, Yu HH, Swager TM, Griffin RG (2004) Dynamic nuclear polarization with biradicals. J Am Chem Soc 126:10844–10845CrossRefGoogle Scholar
  21. Hu KN, Song C, Yu HH, Swager TM, Griffin RG (2008) High-frequency dynamic nuclear polarization using biradicals: a multifrequency EPR lineshape analysis. J Chem Phys 128:052302ADSCrossRefGoogle Scholar
  22. Hu FH, Luo WB, Cady SD, Hong M (2011) Conformational plasticity of the influenza A M2 transmembrane helix in lipid bilayers under varying pH, drug binding, and membrane thickness. Biochim Biophys Acta 1808:415–423CrossRefGoogle Scholar
  23. Jacso T, Franks WT, Rose H, Fink U, Broecker J, Keller S, Oschkinat H, Reif B (2012) Characterization of membrane proteins in isolated native cellular membranes by dynamic nuclear polarization solid-state NMR spectroscopy without purification and reconstitution. Angew Chem Int Ed Engl 51:432–435CrossRefGoogle Scholar
  24. Joh NH, Wang T, Bhate MP, Acharya R, Wu Y, Grabe M, Hong M, Grigoryan G, DeGrado WF (2014) De novo design of a transmembrane Zn2+-transporting four-helix bundle. Science 346:1520–1524ADSCrossRefGoogle Scholar
  25. Koers EJ, van der Cruijsen EAW, Rosay M, Weingarth M, Prokofyev A, Sauvee C, Ouari O, van der Zwan J, Pongs O, Tordo P, Maas WE, Baldus M (2014) NMR-based structural biology enhanced by dynamic nuclear polarization at high magnetic field. J Biomol NMR 60:157–168CrossRefGoogle Scholar
  26. Kosen PA (1989) Spin labeling of proteins. Methods Enzymol 177:86–121CrossRefGoogle Scholar
  27. Kubicki DJ, Casano G, Schwarzwalder M, Abel S, Sauvee C, Ganesan K, Yulikov M, Rossini AJ, Jeschke G, Coperet C, Lesage A, Tordo P, Ouari O, Emsley L (2016) Rational design of dinitroxide biradicals for efficient cross-effect dynamic nuclear polarization. Chem Sci 7:550–558CrossRefGoogle Scholar
  28. Kucerka N, Liu YF, Chu NJ, Petrache HI, Tristram-Nagle ST, Nagle JF (2005) Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles. Biophys J 88:2626–2637CrossRefGoogle Scholar
  29. Lee M, Hong M (2014) Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature. J Biomol NMR 59:263–277CrossRefGoogle Scholar
  30. Linden AH, Lange S, Franks WT, Akbey U, Specker E, van Rossum BJ, Oschkinat H (2011) Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR. J Am Chem Soc 133:19266–19269CrossRefGoogle Scholar
  31. Luo W, Cady SD, Hong M (2009) Immobilization of the influenza A M2 transmembrane peptide in virus-envelope mimetic lipid membranes: a solid-state NMR investigation. Biochemistry 48:6361–6368CrossRefGoogle Scholar
  32. 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 USA 105:883–888ADSCrossRefGoogle Scholar
  33. Maly T, Debelouchina GT, Bajaj VS, Hu KN, Joo CG, Mak-Jurkauskas ML, Sirigiri JR, van der Wel PCA, Herzfeld J, Temkin RJ, Griffin RG (2008) Dynamic nuclear polarization at high magnetic fields. J Chem Phys 128:052211ADSCrossRefGoogle Scholar
  34. Matsuki Y, Maly T, Ouari O, Karoui H, Le Moigne F, Rizzato E, Lyubenova S, Herzfeld J, Prisner T, Tordo P, Griffin RG (2009) Dynamic nuclear polarization with a rigid biradical. Angew Chem Int Ed Engl 48:4996–5000CrossRefGoogle Scholar
  35. Mentink-Vigier F, Paul S, Lee D, Feintuch A, Hediger S, Vega S, De Paëpe G (2015) Nuclear depolarization and absolute sensitivity in magic-angle spinning cross effect dynamic nuclear polarization. Phys Chem Chem Phys 17:21824–21836CrossRefGoogle Scholar
  36. Michaelis VK, Ong TC, Kiesewetter MK, Frantz DK, Walish JJ, Ravera E, Luchinat C, Swager TM, Griffin RG (2014) Topical developments in high-field dynamic nuclear polarization. Isr J Chem 54:207–221CrossRefGoogle Scholar
  37. Nadaud PS, Helmus JJ, Hofer N, Jaroniec CP (2007) Long-range structural restraints in spin-labeled proteins probed by solid-state nuclear magnetic resonance spectroscopy. J Am Chem Soc 129:7502–7503CrossRefGoogle Scholar
  38. Nagle JF, Tristram-Nagle S (2000) Lipid bilayer structure. Curr Opin Struct Biol 10:474–480CrossRefGoogle Scholar
  39. Ni QZ, Daviso E, Can TV, Markhasin E, Jawla SK, Swager TM, Temkin RJ, Herzfeld J, Griffin RG (2013) High frequency dynamic nuclear polarization. Acc Chem Res 46:1933–1941CrossRefGoogle Scholar
  40. Reggie L, Lopez JJ, Collinson I, Glaubitz C, Lorch M (2011) Dynamic nuclear polarization-enhanced solid-state NMR of a 13C-labeled signal peptide bound to lipid-reconstituted Sec translocon. J Am Chem Soc 133:19084–19086CrossRefGoogle Scholar
  41. Renault M, Pawsey S, Bos MP, Koers EJ, Nand D, Tommassen-van Boxtel R, Rosay M, Tommassen J, Maas WE, Baldus M (2012) Solid-state NMR spectroscopy on cellular preparations enhanced by dynamic nuclear polarization. Angew Chem Int Ed Engl 51:2998–3001CrossRefGoogle Scholar
  42. Rosay M, Tometich L, Pawsey S, Bader R, Schauwecker R, Blank M, Borchard PM, Cauffman SR, Felch KL, Weber RT, Temkin RJ, Griffin RG, Maas WE (2010) Solid-state dynamic nuclear polarization at 263 GHz: spectrometer design and experimental results. Phys Chem Chem Phys 12:5850–5860CrossRefGoogle Scholar
  43. Rossini AJ, Zagdoun A, Lelli M, Lesage A, Coperet C, Emsley L (2013) Dynamic nuclear polarization surface enhanced NMR spectroscopy. Acc Chem Res 46:1942–1951CrossRefGoogle Scholar
  44. Salnikov E, Rosay M, Pawsey S, Ouari O, Tordo P, Bechinger B (2010) Solid-state NMR spectroscopy of oriented membrane polypeptides at 100 K with signal enhancement by dynamic nuclear polarization. J Am Chem Soc 132:5940–5941CrossRefGoogle Scholar
  45. Sauvee C, Rosay M, Casano G, Aussenac F, Weber RT, Ouari O, Tordo P (2013) Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency. Angew Chem Int Ed Engl 52:10858–10861CrossRefGoogle Scholar
  46. Sergeyev IV, Day LA, Goldbourt A, McDermott AE (2011) Chemical shifts for the unusual DNA structure in Pf1 bacteriophage from dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy. J Am Chem Soc 133:20208–20217CrossRefGoogle Scholar
  47. Smith AN, Caporini MA, Fanucci GE, Long JR (2015) A method for dynamic nuclear polarization enhancement of membrane proteins. Angew Chem Int Ed Engl 54:1542–1546CrossRefGoogle Scholar
  48. Solomon I (1955) Relaxation processes in a system of 2 spins. Phys Rev 99:559–565ADSCrossRefGoogle Scholar
  49. Song CS, Hu KN, Joo CG, Swager TM, Griffin RG (2006) TOTAPOL: a biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media. J Am Chem Soc 128:11385–11390CrossRefGoogle Scholar
  50. Takahashi H, Lee D, Dubois L, Bardet M, Hediger S, De Paepe G (2012) Rapid natural-abundance 2D 13C–13C correlation spectroscopy using dynamic nuclear polarization enhanced solid-state NMR and matrix-free sample preparation. Angew Chem Int Ed Engl 51:11766–11769CrossRefGoogle Scholar
  51. Thurber KR, Tycko R (2014) Perturbation of nuclear spin polarizations in solid state NMR of nitroxide-doped samples by magic-angle spinning without microwaves. J Chem Phys 140:184201ADSCrossRefGoogle Scholar
  52. van der Cruijsen EAW, Koers EJ, Sauvee C, Hulse RE, Weingarth M, Ouari O, Perozo E, Tordo P, Baldus M (2015) Biomolecular DNP-supported NMR spectroscopy using site-directed spin labeling. Chem Eur J 21:12971–12977CrossRefGoogle Scholar
  53. Voinov MA, Good DB, Ward ME, Milikisiyants S, Marek A, Caporini MA, Rosay M, Munro RA, Ljumovic M, Brown LS, Ladizhansky V, Smirnov AI (2015) Cysteine-specific labeling of proteins with a nitroxide biradical for dynamic nuclear polarization NMR. J Phys Chem B 119:10180–10190CrossRefGoogle Scholar
  54. White SH, Wimley WC (1999) Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct 28:319–365CrossRefGoogle Scholar
  55. Wylie BJ, Dzikovski BG, Pawsey S, Caporini M, Rosay M, Freed JH, McDermott AE (2015) Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces. J Biomol NMR 61:361–367CrossRefGoogle Scholar
  56. Yu ZW, Quinn PJ (1998) The modulation of membrane structure and stability by dimethyl sulphoxide (review). Mol Membr Biol 15:59–68CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Shu Y. Liao
    • 1
  • Myungwoon Lee
    • 1
  • Tuo Wang
    • 1
  • Ivan V. Sergeyev
    • 2
  • Mei Hong
    • 1
  1. 1.Department of ChemistryMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Bruker BiospinBillericaUSA

Personalised recommendations