Skip to main content
SpringerLink
Log in

Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

CP:

Cross polarization

DMPC:

1,2-dimyristoyl-sn-glycero-3-phosphocholine

DHPC:

1,2-dihexanoyl-sn-glycero-3-phosphocholine

MMHH:

Mismatched Hartmann-Hahn Conditions

OS:

Oriented sample

PISEMA:

Polarization inversion spin exchange at the magic angle

PRE:

Paramagnetic relaxation enhancement

REP-CP:

Repetitive cross-polarization

References

  • Abramov G, Morag O, Goldbourt A (2015) Magic-angle spinning NMR of intact bacteriophages: Insights into the capsid, DNA and their interface. J Magn Reson 253:80–90

    Article  ADS  Google Scholar 

  • Al-Abdul-Wahid MS, Neale C, Pomes R, Prosser RS (2009) A Solution NMR Approach to the Measurement of Amphiphile Immersion Depth and Orientation in Membrane Model Systems. J Am Chem Soc 131(18):6452–6459

    Article  Google Scholar 

  • Al-Abdul-Wahid MS, Verardi R, Veglia G, Prosser RS (2011) Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen. J Biomol Nmr 51(1–2):173–183

    Article  Google Scholar 

  • Blumberg WE (1960) Nuclear spin-lattice relaxation caused by paramagnetic impurities. Phys Rev 119(1):79–84

    Article  ADS  Google Scholar 

  • Bruschweiler R, Zhang FL (2004) Covariance nuclear magnetic resonance spectroscopy. J Chem Phys 120(11):5253–5260

    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(4):2363–2373

    Article  Google Scholar 

  • Cavanagh J, Fairbrother WJ, Palmer AG, Skelton NJ (1996) Protein NMR spectroscopy: principles & practice. Academic Press, San Diego

    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(1):89–94

    Article  ADS  Google Scholar 

  • De Angelis AA, Howell SC, Nevzorov AA, Opella SJ (2006) Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy. J Am Chem Soc 128(37):12256–12267

    Article  Google Scholar 

  • Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293

    Article  Google Scholar 

  • Durr, U. H. N., Gildenberg M, Ramamoorthy A (2012) The magic of bicelles lights up membrane protein structure. Chem Rev 112(11):6054–6074

    Article  Google Scholar 

  • Gayen A, Banigan JR, Traaseth NJ (2013) Ligand-induced conformational changes of the multidrug resistance transporter EmrE probed by oriented solid-state NMR spectroscopy. Angew Chem Int Edit 52(39):10321–10324

    Article  Google Scholar 

  • Gerstein BC, Dybowski CR (1985) Transient techniques in NMR of solids: an introduction to theory and practice. Academic Press, New York

    Google Scholar 

  • Gillespie JR, Shortle D (1997) Characterization of long-range structure in the denatured state of staphylococcal nuclease 0.1. Paramagnetic relaxation enhancement by nitroxide spin labels. J Mol Biol 268(1):158–169

    Article  Google Scholar 

  • Gleason NJ, Vostrikov VV, Greathouse DV, Koeppe RE (2013) Buried lysine, but not arginine, titrates and alters transmembrane helix tilt. Proc Natl Acad Sci USA 110(5):1692–1695

    Article  ADS  Google Scholar 

  • Glover KJ, Whiles JA, Wu G, Yu N-J, Deems R, Struppe JO, Stark RE, Komives EA, Vold RR (2001) Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophys J 81:2163–2171

    Article  Google Scholar 

  • Goldbourt A, Gross BJ, Day LA, McDermott AE (2007) Filamentous phage studied by magic-angle spinning NMR: resonance assignment and secondary structure of the coat protein in Pf1. J Am Chem Soc 129(8):2338–2344

    Article  Google Scholar 

  • Gonzalez A, Nave C, Marvin DA (1995) Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 Å resolution X-ray fibre diffraction data. Acta Crystallogr D Biol Crystallogr 51:792–804

    Article  Google Scholar 

  • Jeschke G, Grossmann G (1993) Spinning-side-band-pattern deviations in cross-polarization MAS NMR-spectra. J Magn Reson Ser A 103(3):323–328

    Article  ADS  Google Scholar 

  • Johnson RL, Schmidt-Rohr K (2014) Quantitative solid-state C-13 NMR with signal enhancement by multiple cross polarization. J Magn Reson 239:44–49

    Article  ADS  Google Scholar 

  • Knox RW, Lu J, Opella SJ, Nevzorov AA (2010) A resonance assignment method for oriented sample solid-state NMR of proteins. J Am Chem Soc 132:8255–8257

    Article  Google Scholar 

  • Koroloff SN, Nevzorov AA (2015) Optimization of cross-polarization at low radiofrequency fields for sensitivity enhancement in solid-state NMR of membrane proteins reconstituted in magnetically aligned bicelles. J Magn Reson 256:14–22

    Article  ADS  Google Scholar 

  • Levitt M (1991) Heteronuclear cross polarization in liquid-state nuclear magnetic resonance: mismatch compensation and relaxation behavior. J Chem Phys 94(1):30–38

    Article  ADS  Google Scholar 

  • Linser R, Chevelkov V, Diehl A, Reif B (2007) Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins. J Magn Reson 189(2):209–216

    Article  ADS  Google Scholar 

  • Lu GJ, Son WS, Opella SJ (2011) A general assignment method for oriented sample (OS) solid-state NMR of proteins based on the correlation of resonances through heteronuclear dipolar couplings in samples aligned parallel and perpendicular to the magnetic field. J Magn Reson 209(2):195–206

    Article  ADS  Google Scholar 

  • Marassi FM, Opella SJ (2003) Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints. Prot. Sci. 12(3):403–411

    Article  Google Scholar 

  • McDonnell PA, Shon K, Kim Y, Opella SJ (1993) Fd coat protein structure in membrane environments. J Mol Biol 233(3):447–463

    Article  Google Scholar 

  • Mote KR, Gopinath T, Traaseth NJ, Kitchen J, Gor’kov PL, Brey WW, Veglia G (2011) Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly N-15 labeled integral membrane proteins in magnetically aligned lipid bilayers. J Biomol NMR 51(3):339–346

    Article  Google Scholar 

  • Nadaud PS, Sengupta I, Helmus JJ, Jaroniec CP (2011) Evaluation of the influence of intermolecular electron-nucleus couplings and intrinsic metal binding sites on the measurement of N-15 longitudinal paramagnetic relaxation enhancements in proteins by solid-state NMR. J Biomol Nmr 51(3):293–302

    Article  Google Scholar 

  • Nevzorov AA (2008) Mismatched Hartmann-Hahn conditions cause proton-mediated intermolecular magnetization transfer between dilute low spin nuclei in NMR of static solids. J Am Chem Soc 130:11282–11283

    Article  Google Scholar 

  • Nevzorov AA (2009) High-resolution local-field spectroscopy with internuclear correlations. J Magn Reson 201(1):111–114

    Article  ADS  Google Scholar 

  • Nevzorov AA (2011) Orientational and motional narrowing of solid-state NMR lineshapes of uniaxially aligned membrane proteins. J Phys Chem B 115(51):15406–15414

    Article  Google Scholar 

  • Opella SJ, Marassi FM, Gesell JJ, Valente AP, Kim Y, Oblatt-Montal M, Montal M (1999) Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy. Nat Struct Biol 6(4):374–379

    Article  Google Scholar 

  • Park SH, Mrse AA, Nevzorov AA, De Angelis AA, Opella SJ (2006a) Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy. J Magn Reson 178(1):162–165

    Article  ADS  Google Scholar 

  • Park SH, De Angelis AA, Nevzorov AA, Wu CH, Opella SJ (2006b) Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles. Biophys J 91:3032–3042

    Article  Google Scholar 

  • Park SH, Loudet C, Marassi FM, Dufourc EJ, Opella SJ (2008) Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field. J Magn Reson 193(1):133–138

    Article  ADS  Google Scholar 

  • Park SH, Marassi FM, Black D, Opella SJ (2010) Structure and dynamics of the membrane-bound form of Pf1 coat protein: implications of structural rearrangement for virus assembly. Biophys J 99(5):1465–1474

    Article  Google Scholar 

  • Prosser RS, Luchette PA (2004) An NMR study of the origin of dioxygen-induced spin-lattice relaxation enhancement and chemical shift perturbation. J Magn Reson 171(2):225–232

    Article  ADS  Google Scholar 

  • Prosser RS, Hwang JS, Vold RR (1998) Magnetically aligned phospholipid bilayers with positive ordering: a new model membrane system. Biophys J 74:2405–2418

    Article  Google Scholar 

  • Raya J, Perrone B, Hirschinger J (2013) Chemical shift powder spectra enhanced by multiple-contact cross-polarization under slow magic-angle spinning. J Magn Reson 227:93–102

    Article  ADS  Google Scholar 

  • Sanders CR, Prestegard JH (1990) Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO. Biophys J 58:447–460

    Article  Google Scholar 

  • Sanders CR, Schwonek JP (1992) Characterization of magnetically orientable bilayers in mixtures of DHPC and DMPC by solid state NMR. Biochem. 31:8898–8905

    Article  Google Scholar 

  • Scholz F, Boroske E, Helfrich W (1984) Magnetic-anisotropy of lecithin membranes—a new anisotropy susceptometer. Biophys J 45(3):589–592

    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(50):20208–20217

    Article  Google Scholar 

  • Sharma M, Yi MG, Dong H, Qin HJ, Peterson E, Busath DD, Zhou HX, Cross TA (2010) Insight into the mechanism of the influenza a proton channel from a structure in a lipid bilayer. Science 330(6003):509–512

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  • Tang WX, Nevzorov AA (2011) Repetitive cross-polarization contacts via equilibration-re-equilibration of the proton bath: Sensitivity enhancement for NMR of membrane proteins reconstituted in magnetically aligned bicelles. J Magn Reson 212(1):245–248

    Article  ADS  Google Scholar 

  • Tang WX, Knox RW, Nevzorov AA (2012) A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles. Journal of Biomolecular Nmr 54(3):307–316

    Article  Google Scholar 

  • Tesch DM, Nevzorov AA (2014) Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins. J Magn Reson 239:9–15

    Article  ADS  Google Scholar 

  • Theillet FX, Binolfi A, Liokatis S, Verzini S, Selenko P (2011) Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins. J Biomol Nmr 51(4):487–495

    Article  Google Scholar 

  • Thiriot DS, Nevzorov AA, Zagyanskiy L, Wu CH, Opella SJ (2004) Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy. J Mol Biol 341:869–879

    Article  Google Scholar 

  • Thiriot DS, Nevzorov AA, Opella SJ (2005) Structural basis of the temperature transition of Pf1 bacteriophage. Protein Sci 14:1064–1070

    Article  Google Scholar 

  • Traaseth NJ, Buffy JJ, Zamoon J, Veglia G (2006) Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR. BioChemistry 45(46):13827–13834

    Article  Google Scholar 

  • Traaseth NJ, Ha KN, Verardi R, Shi L, Buffy JJ, Masterson LR, Veglia G (2008) Structural and dynamic basis of phospholamban and sarcolipin inhibition of Ca2+-ATPaset. BioChemistry 47(1):3–13

    Article  Google Scholar 

  • Traaseth NJ, Shi L, Verardi R, Mullen DG, Barany G, Veglia G (2009) Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach. Proc Natl Acad Sci USA 106(25):10165–10170

    Article  ADS  Google Scholar 

  • Verardi R, Shi L, Traaseth NJ, Walsh N, Veglia G (2011) Structural topology of phospholamban pentamer in lipid bilayers by a hybrid solution and solid-state NMR method. Proc Natl Acad Sci USA 108(22):9101–9106

    Article  ADS  Google Scholar 

  • Vold RR, Prosser RS (1996) Magnetically oriented phospholipid bilayered micelles for structural studies of polypeptides. Does the ideal bicelle exist? J Magn Reson B 113:267–271

    Article  Google Scholar 

  • Wang J, Kim S, Kovacs F, Cross TA (2001). Structure of the transmembrane region of the M2 protein H + channel. Prot. Sci. 10:2241–2250

    Article  Google Scholar 

  • Welsh LC, Marvin DA, Perham RN (1998) Analysis of X-ray diffraction from fibres of Pf1 Inovirus (filamentous bacteriophage) shows that the DNA in the virion is not highly ordered. J Mol Biol 284(5):1265–1271

    Article  Google Scholar 

  • Wickramasinghe NP, Shaibat M, Ishii Y (2005) Enhanced sensitivity and resolution in H-1 solid-state NMR spectroscopy of paramagnetic complexes under very fast magic angle spinning. J Am Chem Soc 127(16):5796–5797

    Article  Google Scholar 

  • Wickramasinghe NP, Kotecha M, Samoson A, Past J, Ishii Y (2007) Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation. J Magn Reson 184(2):350–356

    Article  ADS  Google Scholar 

  • Wickramasinghe NP, Parthasarathy S, Jones CR, Bhardwaj C, Long F, Kotecha M, Mehboob S, Fung LWM, Past J, Samoson A, Ishii Y (2009) Nanomole-scale protein solid-state NMR by breaking intrinsic H-1 T-1 boundaries. Nat Methods 6(3):215–218

    Article  Google Scholar 

  • Wu CH, Ramamoorthy A, Opella SJ (1994) High-resolution heteronuclear dipolar solid-state NMR spectroscopy. J Magn Reson A 109:270–272

    Article  ADS  Google Scholar 

  • Xue Y, Podkorytov IS, Rao DK, Benjamin N, Sun HL, Skrynnikov NR (2009) Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain. Protein Sci 18(7):1401–1424

    Article  Google Scholar 

  • Yamamoto K, Xu JD, Kawulka KE, Vederas JC, Ramamoorthy A (2010) Use of a copper-chelated lipid speeds up NMR measurements from membrane proteins. Journal of the American Chemical Society 132(20): 6929–6931

  • Yamamoto K, Gildenberg M, Ahuja S, Im SC, Pearcy P, Waskell L, Ramamoorthy A (2013) Probing the transmembrane structure and topology of microsomal cytochrome-P450 by solid-state NMR on temperature-resistant bicelles. Sci Rep 3(2556):1–6

    Google Scholar 

  • Zhang SM, Wu XL, Mehring M (1990) Successive polarization under mismatched Hartmann-Hahn condition. Chem Phys Lett 166(1):92–94

    Article  ADS  Google Scholar 

Download references

Acknowledgements

Supported by the National Science Foundation.

Author information

Author notes

  1. Deanna M. Tesch

    Present address: Shaw University, 118 E. South Street, Raleigh, NC, 27601, USA

Authors and Affiliations

  1. Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh, NC, 27695-8204, USA

    Sophie N. Koroloff, Deanna M. Tesch, Emmanuel O. Awosanya & Alexander A. Nevzorov

Authors
  1. Sophie N. Koroloff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Deanna M. Tesch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Emmanuel O. Awosanya
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander A. Nevzorov
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Alexander A. Nevzorov.

Additional information

Sophie N. Koroloff and Deanna M. Tesch have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 377 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Koroloff, S.N., Tesch, D.M., Awosanya, E.O. et al. Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals. J Biomol NMR 67, 135–144 (2017). https://doi.org/10.1007/s10858-017-0090-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-017-0090-0

Keywords

Search

Navigation