Abstract
TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the 13C(t 1) dimension with inverted phase corresponding to 1HN resonance frequencies, with approximately 5% intensity of the parent 13C crosspeaks. These artifacts originate from the modulation of the 1HN frequency onto the resonance frequency of 13Cα and/or 13Cβ and are due to 180° pulses imperfections used for 1H decoupling during the 13C(t 1) evolution period. These sidebands can become severe for CAi, CAi−1 and/or CBi, CBi−1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 1H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.
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References
Bodenhausen G, Ruben DJ (1980) Natural abundance N-15 NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185–189
Eletsky A, Kienhofer A, Pervushin K (2001) TROSY NMR with partially deuterated proteins. J Biomol NMR 20:177–180
Emsley L, Bodenhausen G (1992) Optimization of shaped selective pulses for NMR using a quaternion description of their overall propagators. J Magn Reson 97:135–148
Grzesiek S, Bax A (1993) The importance of not saturating H2o in protein NMR - application to sensitivity enhancement and NOE measurements. J Am Chem Soc 115:12593–12594
Levitt MH (2007) Composite pulses in eMagRes. Wiley, Hoboken
Loria JP, Rance M, Palmer AG (1999) A TROSY CPMG sequence for characterizing chemical exchange in large proteins. J Biomol NMR 15:151–155
Pervushin K, Riek R, Wider G, Wuthrich 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
Salzmann M, Pervushin K, Wider G, Senn H, Wuthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci USA 95:13585–13590
Salzmann M, Wider G, Pervushin K, Senn H, Wuthrich K (1999) TROSY-type triple-resonance experiments for sequential NMR assignments of large proteins. J Am Chem Soc 121:844–848
Smith MA, Hu H, Shaka AJ (2001) Improved broadband inversion performance for NMR in liquids. J Magn Reson 151:269–283
Solyom Z et al (2013) BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J Biomol NMR 55:311–321
Wang YX et al (1999) Measurement of (3 h)J(NC ‘) connectivities across hydrogen bonds in a 30 kDa protein. J Biomol NMR 14:181–184
Weigelt J (1998) Single scan, sensitivity- and gradient-enhanced TROSY for multidimensional NMR experiments (vol 120, pg 10778, 1998). J Am Chem Soc 120:12706–12706
Wu P.S.C., Otting G (2005) Rapid pulse length determination in high-resolution NMR. J Magn Reson 176:115–119
Yang DW, Kay LE (1999a) Improved (HN)-H-1-detected triple resonance TROSY-based experiments. J Biomol NMR 13:3–10
Yang DW, Kay LE (1999b) TROSY triple-resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J Am Chem Soc 121:2571–2575
Zhu G, Xia YL, Nicholson LK, Sze KH (2000) Protein dynamics measurements by TROSY-based NMR experiments. J Magn Reson 143:423–426
Acknowledgements
This work is financially supported by the NIH grants GM 100310 to G. V. and AI094623 to C.G.K.. The experiments were carried out at the Minnesota NMR Center (MNMR) and at the National Magnetic Resonance Facility at Madison (NMRFAM) [NIH support: P41GM103399 (formerly P41RR002301); P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, S10RR029220. NSF support: DMB-8415048, OIA-9977486, BIR-9214394]. Many thanks to Prof. E. Komives and Dr. T. Kromann-Tofting at UCSD for providing testing sample.
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Youlin Xia and Paolo Rossi have contributed equally to the work.
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Xia, Y., Rossi, P., Tonelli, M. et al. Optimization of 1H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments. J Biomol NMR 69, 45–52 (2017). https://doi.org/10.1007/s10858-017-0133-6
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DOI: https://doi.org/10.1007/s10858-017-0133-6