Abstract
Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the “shotgun” approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the 15N chemical shift and 15N–1H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an 15N–15N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled “seed” spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B 1 fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.
Similar content being viewed by others
Abbreviations
- DMPC:
-
1,2-Dimyristoyl-sn-glycero-3-phosphocholine
- DHPC:
-
1,2-Dihexanoyl-sn-glycero-3-phosphocholine
- 6-O-PC:
-
1,2-Di-O-hexyl-sn-glycero-3-phosphocholine
- SAMPI4:
-
SAMMY pulse sequence with π/4 pulse correction
- PISA:
-
Polarity index slant angles
- REP-CP:
-
Repetitive cross-polarization
- MMHH:
-
Mismatched Hartmann-Hahn
- PDSD:
-
Proton-driven spin diffusion
References
Aussenac F, Lavigne B, Dufourc EJ (2005) Toward bicelle stability with ether-linked phospholipids: temperature, composition, and hydration diagrams by H-2 and P-31 solid-state NMR. Langmuir 21(16):7129–7135
Cady SD, Hong M (2009) Effects of amantadine on the dynamics of membrane-bound influenza A M2 transmembrane peptide studied by NMR relaxation. J Biomol NMR 45(1–2):185–196
De Angelis AA, Opella SJ (2007) Bicelle samples for solid-state NMR of membrane proteins. Nat Protoc 2(10):2332–2338
De Angelis AA, Howell SC, Opella SJ (2006) Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts. J Magn Reson 183(2):329–332
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(3):277–293
Glover KJ, Whiles JA, Wu GH, Yu NJ, 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(4):2163–2171
Khitrin AK, Xu JD, Ramamoorthy A (2011) Cross-correlations between low-gamma nuclei in solids via a common dipolar bath. J Magn Reson 212(1):95–101
Knox RW, Lu GJ, Opella SJ, Nevzorov AA (2010) A resonance assignment method for oriented-sample solid-state NMR of proteins. J Am Chem Soc 132(24):8255–8257
Lewandowski JR, De Paepe G, Griffin RG (2007) Proton assisted insensitive nuclei cross polarization. J Am Chem Soc 129(4):728–729
Lin EC, Opella SJ (2011) (1)H assisted (13)C/(15)N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples. J Magn Reson 211(1):37–44
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
Marassi FM (2001) A simple approach to membrane protein secondary structure and topology based on NMR spectroscopy. Biophys J 80(2):994–1003
Marassi FM, Opella SJ (2000) A solid-state NMR index of helical membrane protein structure and topology. J Magn Reson 144(1):150–155
Marassi FM, Gesell JJ, Valente AP, Kim Y, Oblatt-Montal M, Montal M, Opella SJ (1999) Dilute spin-exchange assignment of solid-state NMR spectra of oriented proteins: acetylcholine M2 in bilayers. J Biomol NMR 14(2):141–148
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 (15)N labeled integral membrane proteins in magnetically aligned lipid bilayers. J Biomol NMR 51(3):339–346
Nambudripad R, Stark W, Opella SJ, Makowski L (1991) Membrane-mediated assembly of filamentous bacteriophage Pf1 coat protein. Science 252(5010):1305–1308
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(34):11282–11283
Nevzorov AA (2009) High-resolution local field spectroscopy with internuclear correlations. J Magn Reson 201(1):111–114
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
Nevzorov AA, Opella SJ (2007) Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples. J Magn Reson 185(1):59–70
Opella SJ, Zeri AC, Park SH (2008) Structure, dynamics, and assembly of filamentous bacteriophages by nuclear magnetic resonance spectroscopy. Annu Rev Phys Chem 59:635–657
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
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(8):3032–3042
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
Prosser RS, Hunt SA, DiNatale JA, Vold RR (1996) Magnetically aligned membrane model systems with positive order parameter: switching the sign of S-zz with paramagnetic ions. J Am Chem Soc 118(1):269–270
Sanders CR, Hare BJ, Howard KP, Prestegard JH (1994) Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules. Prog Nucl Magn Reson Spectrosc 26:421–444
Suter D, Ernst RR (1985) Spin diffusion in resolved solid-state NMR-spectra. Phys Rev B 32(9):5608–5627
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
Thiriot DS, Nevzorov AA, Opella SJ (2005) Structural basis of the temperature transition of Pf1 bacteriophage. Protein Sci 14(4):1064–1070
Traaseth NJ, Gopinath T, Veglia G (2010) On the performance of spin diffusion NMR techniques in oriented solids: prospects for resonance assignments and distance measurements from separated local field experiments. J Phys Chem B 114(43):13872–13880
Wang J, Denny J, Tian C, Kim S, Mo Y, Kovacs F, Song Z, Nishimura K, Gan Z, Fu R, Quine JR, Cross TA (2000) Imaging membrane protein helical wheels. J Magn Reson 144(1):162–167
Xu JD, Smith PES, Soong R, Ramamoorthy A (2011) A proton spin diffusion based solid-state NMR approach for structural studies on aligned samples. J Phys Chem B 115(16):4863–4871
Acknowledgments
We would like to thank Prof. Stanley J. Opella and George J. Lu (UCSD) for stimulating discussions. Research supported by a grant from NSF (MCB 0843520).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Tang, W., Knox, R.W. & Nevzorov, A.A. A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles. J Biomol NMR 54, 307–316 (2012). https://doi.org/10.1007/s10858-012-9673-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10858-012-9673-y