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Journal of Biomolecular NMR

, Volume 61, Issue 3–4, pp 361–367 | Cite as

Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces

  • Benjamin J. Wylie
  • Boris G. Dzikovski
  • Shane Pawsey
  • Marc Caporini
  • Melanie Rosay
  • Jack H. Freed
  • Ann E. McDermott
Article

Abstract

We demonstrate that dynamic nuclear polarization of membrane proteins in lipid bilayers may be achieved using a novel polarizing agent: pairs of spin labels covalently bound to a protein of interest interacting at an intermolecular interaction surface. For gramicidin A, nitroxide tags attached to the N-terminal intermolecular interface region become proximal only when bimolecular channels forms in the membrane. We obtained signal enhancements of sixfold for the dimeric protein. The enhancement effect was comparable to that of a doubly tagged sample of gramicidin C, with intramolecular spin pairs. This approach could be a powerful and selective means for signal enhancement in membrane proteins, and for recognizing intermolecular interfaces.

Keywords

Dynamic nuclear polarization DNP Membrane proteins Solid-state NMR Gramicidin DEER spectroscopy 

Notes

Acknowledgments

This work was supported by grants from the National Institutes of Health and National Science Foundation. Professor McDermott is a member of the New York Structural Biology Center. The Center is a STAR center supported by the New York State Office of Science, Technology, and Academic Research. NMR resources were supported by NIH P41 GM66354. This work was supported by NSF MCB 0749381, NIH R01 GM 88724 to A. E. M., NIH NRSA F32 087908 to B. J. W. and by NIH/NIGMS P41GM103521 and NIH/NIBIB R01EB003150 to J.H.F.

Supplementary material

10858_2015_9919_MOESM1_ESM.docx (735 kb)
Supplementary material 1 (DOCX 734 kb)

References

  1. 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 189:251–279CrossRefADSGoogle Scholar
  2. 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 106:9244–9249CrossRefADSGoogle Scholar
  3. 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
  4. Becerra LR, Gerfen GJ, Bellew BF, Bryant JA, Hall DA, Inati SJ, Weber RT, Un S, Prisner TF, Mcdermott AE, Fishbein KW, Kreischer KE, Temkin RJ, Singel DJ, Griffin RG (1995) A spectrometer for dynamic nuclear-polarization and electron-paramagnetic-resonance at high-frequencies. J Magn Reson Ser A 117:28–40CrossRefADSGoogle Scholar
  5. Carver TR, Slichter CP (1953) Polarization of nuclear spins in metals. Phys Rev 92:212–213CrossRefADSGoogle Scholar
  6. Dzikovski BG, Borbat PP, Freed JH (2004) Spin-labeled gramicidin A: channel formation and dissociation. Biophys J 87:3504–3517CrossRefGoogle Scholar
  7. Dzikovski BG, Borbat PP, Freed JH (2011) Channel and nonchannel forms of spin-labeled gramicidin in membranes and their equilibria. J Phys Chem B 115:176–185CrossRefGoogle Scholar
  8. Fung BM, Khitrin AK, Ermolaev K (2000) An improved broadband decoupling sequence for liquid crystals and solids. J Magn Reason 142(1):97–101Google Scholar
  9. 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
  10. Hong M, Zhang Y, Hu FH (2012) Membrane protein structure and dynamics from NMR spectroscopy. Annu Rev Phys Chem 63:1–24CrossRefADSGoogle Scholar
  11. Hu KN, Yu HH, Swager TM, Griffin RG (2004) Dynamic nuclear polarization with biradicals. J Am Chem Soc 126:10844–10845CrossRefGoogle Scholar
  12. 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(5):052302(1–17)Google Scholar
  13. Jaroniec CP (2012) Solid-state nuclear magnetic resonance structural studies of proteins using paramagnetic probes. Solid State Nucl Mag 43–44:1–13CrossRefGoogle Scholar
  14. Ketchem RR, Lee KC, Huo S, Cross TA (1996) Macromolecular structural elucidation with solid-state NMR-derived orientational constraints. J Biomol NMR 8:1CrossRefGoogle Scholar
  15. 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
  16. 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
  17. McDermott A (2009) Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Annu Rev Biophys 38:385–403CrossRefMathSciNetGoogle Scholar
  18. 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(24):7502–7503CrossRefGoogle Scholar
  19. Opella SJ, Marassi FM (2004) Structure determination of membrane proteins by NMR spectroscopy. Chem Rev 104:3587–3606CrossRefGoogle Scholar
  20. Pepinsky RB, Feigenson GW (1978) Purification of gramicidin C. Anal Biochem 86(2):512–518CrossRefGoogle Scholar
  21. Quist PO (1998) C-13 solid-state NMR of gramicidin A in a lipid membrane. Biophys J 75:2478–2488CrossRefGoogle Scholar
  22. Rabenstein MD, Shin Y-K (1995) Determination of a distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci USA 92:8239–8243CrossRefADSGoogle Scholar
  23. Rosay M, Lansing JC, Haddad KC, Bachovchin WW, Herzfeld J, Temkin RJ, Griffin RG (2003) High-frequency dynamic nuclear polarization in MAS spectra of membrane and soluble proteins. J Am Chem Soc 125:13626–13627CrossRefGoogle Scholar
  24. Salnikov ES, Ouari O, Koers E, Sarrouj H, Franks T, Rosay M, Pawsey S, Reiter C, Bandara P, Oschkinat H, Tordo P, Engelke F, Bechinger B (2012) Developing DNP/solid-state NMR spectroscopy of oriented membranes. Appl Magn Reson 43:91–106CrossRefGoogle Scholar
  25. 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
  26. Vitzthum V, Borcard F, Jannin S, Morin M, Mieville P, Caporini MA, Sienkiewicz A, Gerber-Lemaire S, Bodenhausen G (2011) Fractional spin-labeling of polymers for enhancing NMR sensitivity by solvent-free dynamic nuclear polarization. ChemPhysChem 12:2929–2932CrossRefGoogle Scholar
  27. Warschawski DE, Arnold AA, Beaugrand M, Gravel A, Chartrand E, Marcotte I (2011) Choosing membrane mimetics for NMR structural studies of transmembrane proteins. BBA Biomembr 1808:1957–1974CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Benjamin J. Wylie
    • 1
  • Boris G. Dzikovski
    • 2
  • Shane Pawsey
    • 3
  • Marc Caporini
    • 3
  • Melanie Rosay
    • 3
  • Jack H. Freed
    • 2
  • Ann E. McDermott
    • 1
  1. 1.Department of ChemistryColumbia UniversityNew YorkUSA
  2. 2.National Biomedical Center for Advanced ESR Technology, Department of Chemistry and Chemical BiologyCornell UniversityIthacaUSA
  3. 3.Bruker BioSpin CorporationBillericaUSA

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