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MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21

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Abstract

We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2TN polarization transfer delay for labeling of 15N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in 15N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)NHs, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161–166, 2005). Therefore, cross peak overlap is diminished while the time period during which the 15N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19–0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.

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References

  • Abrogast L, Majumdar A, Tolman JR (2010) HNCO-based measurement of one-bond amide 15N–1H couplings with optimized precision. J Biomol NMR 46:175–189

    Article  Google Scholar 

  • Andersson P, Annila A, Otting G (1998) An α/β-HSQC-α/β experiment for spin-state selective editing of IS cross peaks. J Magn Reson 133:364–367

    Article  ADS  Google Scholar 

  • Annila A, Permi P (2004) Weakly aligned biological macromolecules in dilute aqueous liquid crystals. Concepts Magn Reson 23A:22–37

    Article  Google Scholar 

  • Bax A, Kontaxis G, Tjandra N (2001) Dipolar couplings in macromolecular structure determination. Methods Enzymol 339:127–174

    Article  Google Scholar 

  • Blackledge M (2005) Recent progress in the study of biomolecular structure and dynamics in solution from residual dipolar couplings. Prog Nucl Magn Reson Spectr 46:23–61

    Article  Google Scholar 

  • Bodenhausen G, Ernst RR (1981) The accordion experiment, a simple approach to 3-dimensional NMR spectroscopy. J Magn Reson 45:367–373

    Google Scholar 

  • Bouvignies G, Markwick PRL, Blackledge M (2007) Simultaneous definition of high resolution protein structure and backbone conformational dynamics using NMR residual dipolar couplings. ChemPhysChem 8:1901–1909

    Article  Google Scholar 

  • Düx P, Whitehead B, Boelens R, Kaptein R, Vuister GW (1999) Measurement of 15N–1H coupling constants in uniformly 15N-labeled proteins: application to the photoactive yellow protein. J Biomol NMR 10:301–306

    Article  Google Scholar 

  • Fischer MW, Losonczi JA, Weaver JL, Prestegard JH (1999) Domain orientation and dynamics in multidomain protein from residual dipolar couplings. Biochemistry 38:9013–9022

    Article  Google Scholar 

  • Fredriksson K, Louhivuori M, Permi P, Annila A (2004) On the interpretation of residual dipolar couplings as reporters of molecular dynamics. J Am Chem Soc 126:12646–12650

    Article  Google Scholar 

  • Hansen MR, Mueller L, Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol 5:1065–1074

    Article  Google Scholar 

  • Heikkinen S, Aitio H, Permi P, Folmer R, Lappalainen K, Kilpeläinen I (1999) J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHα) couplings in 15N-labeled proteins. J Magn Reson 137:243–246

    Article  ADS  Google Scholar 

  • Heikkinen OK, Ruskamo S, Konarev PV, Svergun DI, Iivanainen T, Heikkinen SM, Permi P, Koskela H, Kilpeläinen I, Ylänne J (2009) Atomic structures of two novel immunoglobulin-like domain pairs in the actin cross-linking protein filamin. J Biol Chem 284:25450–25458

    Article  Google Scholar 

  • Hu K, Doucleff M, Clore GM (2009) Using multiple quantum-coherence to increase the 15N resolution in a three-dimensional TROSY-HNCO experiment for accurate PRE and RDC measurements. J Magn Reson 200:173–177

    Article  ADS  Google Scholar 

  • Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient-enhanced heteronuclear single-quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665

    Article  Google Scholar 

  • Kontaxis G, Clore GM, Bax A (2000) Evaluation of cross-correlation effects and measurement of one-bond couplings in proteins with short transverse relaxation times. J Magn Reson 143:184–196

    Article  ADS  Google Scholar 

  • Lad Y, Kiema T, Jiang P, Pentikäinen O, Coles CH, Campbell ID, Calderwood DA, Ylänne J (2007) Structure of three tandem filamin domains reveals auto-inhibition of ligand binding. EMBO J 26:3993–4004

    Article  Google Scholar 

  • Lakomek NA, Carlomagno T, Becker S, Griesinger C, Meiler J (2006) A thorough dynamic interpretation of residual dipolar couplings in ubiquitin. J Biomol NMR 34:101–115

    Article  Google Scholar 

  • Lerche MH, Meissner A, Poulsen FM, Sørensen OW (1999) Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone. J Magn Reson 140:259–263

    Article  ADS  Google Scholar 

  • Losonczi JA, Andrec M, Fischer MWF, Prestegard JH (1999) Order matrix analysis of residual dipolar couplings using singular-value decomposition. J Magn Reson 138:334–342

    Article  ADS  Google Scholar 

  • Madsen JC, Sørensen OW, Sørensen P, Poulsen FM (1993) Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins. J Biomol NMR 3:239–244

    Article  Google Scholar 

  • Marion D, Ikura M, Tschudin R, Bax A (1989) Rapid recording of 2D NMR-spectra without phase cycling—application to the study of hydrogen-exchange in proteins. J Magn Reson 85:393–399

    Google Scholar 

  • McCoy MA, Mueller L (1992) Selective shaped pulse decoupling in NMR: homonuclear [13C]carbonyl decoupling. J Am Chem Soc 114:2108–2112

    Article  Google Scholar 

  • Meissner A, Duus JO, Sørensen OW (1997) Integration of spin-state-selective excitation into 2D NMR correlation experiments with the heteronuclear ZQ/DQ π rotations for 1JXH-resolved E.COSY-type measurement of heteronuclear coupling constants in proteins. J Biomol NMR 10:89–94

    Article  Google Scholar 

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

    Article  Google Scholar 

  • Ottiger M, Delaglio F, Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378

    Article  ADS  Google Scholar 

  • Pääkkönen K, Sorsa T, Drakenberg T, Pollesello P, Tilgmann C, Permi P, Heikkinen S, Kilpeläinen I, Annila A (2000) Conformations of the regulatory domain of cardiac troponin C examined by residual dipolar couplings. Eur J Biochem 267:6665–6672

    Article  Google Scholar 

  • Permi P (2002) A spin-state-selective experiment for measuring heteronuclear one-bond and homonuclear two-bond couplings from an HSQC-type spectrum. J Biomol NMR 22:27–35

    Article  Google Scholar 

  • Permi P (2003) Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment. J Biomol NMR 27:341–349

    Article  Google Scholar 

  • Permi P, Rosevear PR, Annila A (2000a) A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H) labeled proteins. J Biomol NMR 17:43–54

    Article  Google Scholar 

  • Permi P, Kilpeläinen I, Annila A (2000b) Determination of backbone angle ψ in proteins using a TROSY-based α/β-HN(CO)CA-J experiment. J Magn Reson 146:255–259

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  • Prestegard JH, Al-Hashimi HM, Tolman JR (2000) NMR structures of biomolecules using field oriented media and residual dipolar couplings. Quart Rev Biophys 33:371–424

    Article  Google Scholar 

  • Puttonen E, Tossavainen H, Permi P (2006) Simultaneous determination of one- and two-bond scalar and residual dipolar couplings between 13C′, 13Cα, and 15N spins in proteins. Magn Reson Chem 44:168–176

    Article  Google Scholar 

  • Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1998) TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins. Proc Natl Acad Sci U S A 95:13585–13590

    Article  ADS  Google Scholar 

  • Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114

    Article  ADS  Google Scholar 

  • Tolman JR, Ruan K (2006) NMR residual dipolar couplings as probes of biomolecular dynamics. Chem Rev 106:1720–1736

    Article  Google Scholar 

  • Tugarinov V, Kay LE (2003) Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G. J Mol Biol 327:1121–1133

    Article  Google Scholar 

  • Wang AC, Bax A (1995) Reparametrization of the Karplus relation for 3J(Hα-N) and 3J(HN-C′) in peptides from uniformly 13C/15N-enriched human ubiquitin. J Am Chem Soc 117:1810–1813

    Article  Google Scholar 

  • Weigelt J (1998) Single scan, sensitivity- and gradient-enhanced TROSY for multidimensional NMR experiments. J Am Chem Soc 120:10778–10779

    Article  Google Scholar 

  • Yang DW, Kay LE (1999) Improved 1HN-detected triple resonance TROSY-based experiments. J Biomol NMR 13:3–10

    Article  Google Scholar 

  • Yang DW, Venters RA, Mueller GA, Choy WY, Kay LE (1999) TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N–13CO, 1HN-13CO, 13CO-13Ca and 1HN-13Ca dipolar couplings in 15N, 13C, 2H-labeled proteins. J Biomol NMR 14:333–343

    Article  Google Scholar 

  • Zweckstetter M, Bax A (2000) Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J Am Chem Soc 122:3791–3792

    Article  Google Scholar 

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Authors and Affiliations

  1. NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland

    Sampo Mäntylahti & Perttu Permi

  2. Laboratory of Organic Chemistry, Department of Chemistry, University of Helsinki, P.O. Box 55, 00014, Helsinki, Finland

    Outi Koskela

  3. Biochemistry Department, University of Oxford, Oxford, OX1 3QU, UK

    Pengju Jiang

Authors
  1. Sampo Mäntylahti
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  2. Outi Koskela
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  3. Pengju Jiang
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  4. Perttu Permi
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Correspondence to Perttu Permi.

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Mäntylahti, S., Koskela, O., Jiang, P. et al. MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21. J Biomol NMR 47, 183–194 (2010). https://doi.org/10.1007/s10858-010-9422-z

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  • DOI: https://doi.org/10.1007/s10858-010-9422-z

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