Skip to main content
Log in

A 3D time-shared NOESY experiment designed to provide optimal resolution for accurate assignment of NMR distance restraints in large proteins

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

Abstract

Structure determination of proteins by solution NMR has become an established method, but challenges increase steeply with the size of proteins. Notably, spectral crowding and signal overlap impair the analysis of cross-peaks in NOESY spectra that provide distance restraints for structural models. An optimal spectral resolution can alleviate overlap but requires prohibitively long experimental time with existing methods. Here we present a time-shared 3D experiment optimized for large proteins that provides 15N and 13C dispersed NOESY spectra in a single measurement. NOESY correlations appear in the detected dimension and hence benefit from the highest resolution achievable of all dimensions without increase in experimental time. By design, this experiment is inherently optimal for non-uniform sampling acquisition when compared to current alternatives. Thus, 15N and 13C dispersed NOESY spectra with ultra-high resolution in all dimensions were acquired in parallel within about 4 days instead of 80 days for a 52 kDa monomeric protein at a concentration of 350 μM.

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

Access this article

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

Similar content being viewed by others

References

  • Ayala I, Sounier R, Usé N et al (2009) An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein. J Biomol NMR 43:111–119. doi:10.1007/s10858-008-9294-7

    Article  Google Scholar 

  • Barna JCJ, Laue ED, Mayger MR et al (1987) Exponential sampling, an alternative method for sampling in two-dimensional NMR experiments. J Magn Reson 77:69–77

    ADS  Google Scholar 

  • Bodenhausen G, Ruben DJ (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189

    Article  ADS  Google Scholar 

  • Boelens R, Burgering M, Fogh RH, Kaptein R (1994) Time-saving methods for heteronuclear multidimensional NMR of ((13)C, (15)N) doubly labeled proteins. J Biomol NMR 4:201–213. doi:10.1007/BF00175248

    Article  Google Scholar 

  • Brutscher B, Pardi A, Marion D (1998) Improved sensitivity and resolution in 1 H-13 C NMR experiments of RNA. J Am Chem Soc 120:11845–11851

    Article  Google Scholar 

  • Cai M, Huang Y, Sakaguchi K et al (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J Biomol NMR 11:97–102

    Article  Google Scholar 

  • Coggins BE, Venters RA, Zhou P (2010) Radial sampling for fast NMR: concepts and practices over three decades. Prog Nucl Magn Reson Spectrosc 57:381–419. doi:10.1016/j.pnmrs.2010.07.001

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Farmer BT II (1991) Simultaneous[13C,15 N]-HMQC, a pseudo-triple-resonance experiment. J Magn Reson 93:635–641

    ADS  Google Scholar 

  • Frueh DP, Vosburg DA, Walsh CT, Wagner G (2006) Determination of all nOes in 1H-13C-Me-ILV-U-2H-15 N proteins with two time-shared experiments. J Biomol NMR 34:31–40. doi:10.1007/s10858-005-5338-4

    Article  Google Scholar 

  • Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406. doi:10.1146/annurev.biophys.27.1.357

    Article  Google Scholar 

  • Gelis I, Bonvin AMJJ, Keramisanou D et al (2007) Structural basis for signal sequence recognition by the 204-kDa translocase motor SecA determined by NMR. Cell 131:756–769

    Article  Google Scholar 

  • Goto NK, Kay LE (2000) New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr Opin Struct Biol 10:585–592

    Article  Google Scholar 

  • Goto NK, Gardner KH, Mueller GA et al (1999) A robust and cost-effective method for the production of Val, Leu, Ile (δ1). J Biomol NMR 13:369–374

    Article  Google Scholar 

  • Gross JD, Gelev VM, Wagner G (2003) A sensitive and robust method for obtaining intermolecular NOEs between side chains in large protein complexes. J Biomol NMR 25(3):235–242

    Article  Google Scholar 

  • Guo C, Tugarinov V (2009) Identification of HN-methyl NOEs in large proteins using simultaneous amide-methyl TROSY-based detection. J Biomol NMR 43:21–30. doi:10.1007/s10858-008-9285-8

  • Holland DJ, Bostock MJ, Gladden LF, Nietlispach D (2011) Fast multidimensional NMR spectroscopy using compressed sensing. Angew Chem Int Ed Engl 50:6548–6551. doi:10.1002/anie.201100440

    Article  Google Scholar 

  • Hyberts SG, Milbradt AG, Wagner AB et al (2012) Application of iterative soft thresholding for fast reconstruction of NMR data non-uniformly sampled with multidimensional Poisson Gap scheduling. J Biomol NMR 52:315–327. doi:10.1007/s10858-012-9611-z

    Article  Google Scholar 

  • Hyberts SG, Arthanari H, Robson SA, Wagner G (2014) Perspectives in magnetic resonance: NMR in the post-FFT era. J Magn Reson 241:60–73. doi:10.1016/j.jmr.2013.11.014

    Article  ADS  Google Scholar 

  • Isaacson RL, Simpson PJ, Liu M et al (2007) A new labeling method for methyl transverse relaxation-optimized spectroscopy NMR spectra of alanine residues. J Am Chem Soc 129:15428–15429. doi:10.1021/ja0761784

    Article  Google Scholar 

  • Jee J, Güntert P (2003) Influence of the completeness of chemical shift assignments on NMR structures obtained with automated NOE assignment. J Struct Funct Genomics 4:179–189

    Article  Google Scholar 

  • Kazimierczuk K, Orekhov VY (2011) Accelerated NMR spectroscopy by using compressed sensing. Angew Chem Int Ed Engl 50:5556–5559. doi:10.1002/anie.201100370

    Article  Google Scholar 

  • Keating TA, Miller DA, Walsh CT (2000) Expression, purification, and characterization of HMWP2, a 229 kDa, six domain protein subunit of Yersiniabactin synthetase. Biochemistry 39:4729–4739

    Article  Google Scholar 

  • Keller RLJ (2003) The CARA/Lua programmers manual

  • Maciejewski MW, Mobli M, Schuyler AD et al (2012) Data sampling in multidimensional NMR: Fundamentals and strategies. In: Billiter M, Orekhov V (eds) Novel sampling approaches in higher dimensional NMR: Top Curr Chem, vol 316. Springer, Berlin, pp 49–77. doi:10.1007/128

  • Marion D (2006) Processing of ND NMR spectra sampled in polar coordinates: a simple Fourier transform instead of a reconstruction. J Biomol NMR 36:45–54. doi:10.1007/s10858-006-9066-1

    Article  Google Scholar 

  • Marion D, Wuthrich K (1983) Application of phase sensitive two-dimensional correlated spectroscopy (cosy) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun 113:967–974

    Article  Google Scholar 

  • Meissner A, Sørensen OW (2000) Three-dimensional protein NMR TROSY-type 15 N-resolved 1 HN–1 HN NOESY spectra with diagonal peak suppression. J Magn Reson 142:195–198

    Article  ADS  Google Scholar 

  • Muchmore DC, McIntosh LP, Russell CB, Anderson DE, Dahlquist FW (1989) Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. In: Norman JO, Thomas LJ (eds) Methods in enzymology, vol 177. Academic Press, pp 44–73. doi: 10.1016/0076-6879(10)77005-1

  • Nabuurs SB, Spronk CAEM, Vuister GW, Vriend G (2006) Traditional biomolecular structure determination by NMR spectroscopy allows for major errors. PLoS Comput Biol 2:e9. doi:10.1371/journal.pcbi.0020009

    Article  ADS  Google Scholar 

  • Nietlispach D (2005) Suppression of anti-TROSY lines in a sensitivity enhanced gradient selection TROSY scheme. J Biomol NMR 31:161–166. doi:10.1007/s10858-004-8195-7

    Article  Google Scholar 

  • Orekhov VY, Jaravine VA (2011) Analysis of non-uniformly sampled spectra with multi-dimensional decomposition. Prog Nucl Magn Reson Spectrosc 59:271–292. doi:10.1016/j.pnmrs.2011.02.002

    Article  Google Scholar 

  • Orekhov VY, Ibraghimov I, Billeter M (2003) Optimizing resolution in multidimensional NMR by three-way decomposition. J Biomol NMR 27:165–173

    Article  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371

    Article  ADS  Google Scholar 

  • Pervushin KV, Wider G, Wüthrich K (1998) Single transition-to-single transition polarization transfer (ST2-PT) in [15 N,1H]-TROSY. J Biomol NMR 12:345–348. doi:10.1023/A:1008268930690

    Article  Google Scholar 

  • Piotto M, Saudek V, Sklenár V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665

    Article  Google Scholar 

  • Roehrl MHA, Heffron GJ, Wagner G (2005) Correspondence between spin-dynamic phases and pulse program phases of NMR spectrometers. J Magn Reson 174:325–330. doi:10.1016/j.jmr.2005.02.001

    Article  ADS  Google Scholar 

  • Rovnyak D, Frueh DP, Sastry M et al (2004) Accelerated acquisition of high resolution triple-resonance spectra using non-uniform sampling and maximum entropy reconstruction. J Magn Reson 170:15–21. doi:10.1016/j.jmr.2004.05.016

    Article  ADS  Google Scholar 

  • Sattler M, Maurer M, Schleucher J, Griesinger C (1995) A simultaneous (15)N, (1)H- and (13)C, (1)H-HSQC with sensitivity enhancement and a heteronuclear gradient echo. J Biomol NMR 5:97–102. doi:10.1007/BF00227475

  • Shaka AJ, Reeler J, Frenkiel T, Freeman RAY (1983) An improved sequence for broadband decoupling: WALTZ-16. J Magn Reson 52:335–338

    ADS  Google Scholar 

  • Sinha K, Jen-Jacobson L, Rule GS (2011) Specific labeling of threonine methyl groups for NMR studies of protein–nucleic acid complexes. Biochemistry 50:10189–10191. doi:10.1021/bi201496d

    Article  Google Scholar 

  • Tikole S, Jaravine V, Orekhov VY, Güntert P (2013) Effects of NMR spectral resolution on protein structure calculation. PLoS ONE 8:e68567. doi:10.1371/journal.pone.0068567

    Article  ADS  Google Scholar 

  • Van Ingen H, Vuister GW, Tessari M (2002) A two-dimensional artifact from asynchronous decoupling. J Magn Reson 156:258–261. doi:10.1006/jmre.2002.2564

    Article  ADS  Google Scholar 

  • Würtz P, Aitio O, Hellman M, Permi P (2007) Simultaneous detection of amide and methyl correlations using a time shared NMR experiment: application to binding epitope mapping. J Biomol NMR 39:97–105. doi:10.1007/s10858-007-9178-2

    Article  Google Scholar 

  • Yang D, Kay LE (1999) Improved 1HN-detected triple resonance TROSY-based experiments. J Biomol NMR 13:3–10. doi:10.1023/A:1008329230975

    Article  Google Scholar 

  • Zhu G, Xia Y, Sze KH, Yan X (1999) 2D and 3D TROSY-enhanced NOESY of 15 N labeled proteins. J Biomol NMR 14:377–381

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by NIH, Grant R01-GM104257.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dominique P. Frueh.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 705 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mishra, S.H., Harden, B.J. & Frueh, D.P. A 3D time-shared NOESY experiment designed to provide optimal resolution for accurate assignment of NMR distance restraints in large proteins. J Biomol NMR 60, 265–274 (2014). https://doi.org/10.1007/s10858-014-9873-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-014-9873-8

Keywords

Navigation