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

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Abstract

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.

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References

  • 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 338:181–228

    Article  Google Scholar 

  • 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 52:2774–2782

    Article  Google Scholar 

  • 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 189:251–279

    Article  ADS  Google Scholar 

  • 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 106:9244–9249

    Article  ADS  Google Scholar 

  • 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 34:237–263

    Article  Google Scholar 

  • 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 71:3561–3564

    Article  ADS  Google Scholar 

  • 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 112:9896–9901

    Article  ADS  Google Scholar 

  • Bloembergen N (1957) Proton relaxation times in paramagnetic solutions—comment. J Chem Phys 27:595–596

    Article  ADS  Google Scholar 

  • 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 85:2363–2373

    Article  Google Scholar 

  • 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 133:4274–4284

    Article  Google Scholar 

  • 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 133:11572–11579

    Article  Google Scholar 

  • Can TV, Ni QZ, Griffin RG (2015) Mechanisms of dynamic nuclear polarization in insulating solids. J Magn Reson 253:23–35

    Article  ADS  Google Scholar 

  • Carver TR, Slichter CP (1956) Experimental verification of the overhauser nuclear polarization effect. Phys Rev 102:975–980

    Article  ADS  Google Scholar 

  • Cheng CY, Han SI (2013) Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems. Ann Rev Phys Chem 64:507–532

    Article  ADS  Google Scholar 

  • Chu S, Maltsev S, Emwas AH, Lorigan GA (2010) Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers. J Magn Reson 207:89–94

    Article  ADS  Google Scholar 

  • Craven BM (1976) Crystal-structure of cholesterol monohydrate. Nature 260:727–729

    Article  ADS  Google Scholar 

  • 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 127:12291–12305

    Article  Google Scholar 

  • 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 102:9494–9497

    Article  ADS  Google Scholar 

  • 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 276:930–932

    Article  Google Scholar 

  • Hu KN, Yu HH, Swager TM, Griffin RG (2004) Dynamic nuclear polarization with biradicals. J Am Chem Soc 126:10844–10845

    Article  Google Scholar 

  • 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 128:052302

    Article  ADS  Google Scholar 

  • 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 1808:415–423

    Article  Google Scholar 

  • 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 51:432–435

    Article  Google Scholar 

  • 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 346:1520–1524

    Article  ADS  Google Scholar 

  • 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 60:157–168

    Article  Google Scholar 

  • Kosen PA (1989) Spin labeling of proteins. Methods Enzymol 177:86–121

    Article  Google Scholar 

  • 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 7:550–558

    Article  Google Scholar 

  • 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 88:2626–2637

    Article  Google Scholar 

  • 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 59:263–277

    Article  Google Scholar 

  • 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 133:19266–19269

    Article  Google Scholar 

  • 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 48:6361–6368

    Article  Google Scholar 

  • 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 105:883–888

    Article  ADS  Google Scholar 

  • 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 128:052211

    Article  ADS  Google Scholar 

  • 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 48:4996–5000

    Article  Google Scholar 

  • 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 17:21824–21836

    Article  Google Scholar 

  • 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 54:207–221

    Article  Google Scholar 

  • 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 129:7502–7503

    Article  Google Scholar 

  • Nagle JF, Tristram-Nagle S (2000) Lipid bilayer structure. Curr Opin Struct Biol 10:474–480

    Article  Google Scholar 

  • 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 46:1933–1941

    Article  Google Scholar 

  • 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 133:19084–19086

    Article  Google Scholar 

  • 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 51:2998–3001

    Article  Google Scholar 

  • 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 12:5850–5860

    Article  Google Scholar 

  • Rossini AJ, Zagdoun A, Lelli M, Lesage A, Coperet C, Emsley L (2013) Dynamic nuclear polarization surface enhanced NMR spectroscopy. Acc Chem Res 46:1942–1951

    Article  Google Scholar 

  • 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 132:5940–5941

    Article  Google Scholar 

  • 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 52:10858–10861

    Article  Google Scholar 

  • 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 133:20208–20217

    Article  Google Scholar 

  • Smith AN, Caporini MA, Fanucci GE, Long JR (2015) A method for dynamic nuclear polarization enhancement of membrane proteins. Angew Chem Int Ed Engl 54:1542–1546

    Article  Google Scholar 

  • Solomon I (1955) Relaxation processes in a system of 2 spins. Phys Rev 99:559–565

    Article  ADS  Google Scholar 

  • 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 128:11385–11390

    Article  Google Scholar 

  • 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 51:11766–11769

    Article  Google Scholar 

  • 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 140:184201

    Article  ADS  Google Scholar 

  • 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 21:12971–12977

    Article  Google Scholar 

  • 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 119:10180–10190

    Article  Google Scholar 

  • White SH, Wimley WC (1999) Membrane protein folding and stability: physical principles. Annu Rev Biophys Biomol Struct 28:319–365

    Article  Google Scholar 

  • 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 61:361–367

    Article  Google Scholar 

  • Yu ZW, Quinn PJ (1998) The modulation of membrane structure and stability by dimethyl sulphoxide (review). Mol Membr Biol 15:59–68

    Article  Google Scholar 

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Acknowledgments

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.

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Correspondence to Mei Hong.

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Liao, S.Y., Lee, M., Wang, T. et al. Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR 64, 223–237 (2016). https://doi.org/10.1007/s10858-016-0023-3

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