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Improved accuracy in measuring one-bond and two-bond 15N,13Cα coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

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Abstract

An extension to HN(CO-α/β-N,Cα-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants 1 J N′(i)Cα(i), 2 J HN(i)Cα(i), 2 J Cα(i−1)N′(i), and 3 J Cα(i−1)HN(i) in 15N,13C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the 2 J CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both 2 J CαN′ and 1 J N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.

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Acknowledgments

We thank Bernhard Brutscher, Norman Spitzner, and Marco Betz for providing samples of ubiquitin, RNase T1, and xylanase, respectively. Financial support by the Access to Research Infrastructures activity in the 7th Framework Programme of the EC (Project number: 261863, Bio-NMR) for conducting the research is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Max-von-Laue-Str. 9, 60438, Frankfurt am Main, Germany

    Frank Löhr, Sina Reckel, Susanne Stefer & Volker Dötsch

  2. School of Biosciences, University of Kent, Giles Lane, Canterbury, Kent, CT2 7NJ, UK

    Jürgen M. Schmidt

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  1. Frank Löhr
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  2. Sina Reckel
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  3. Susanne Stefer
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  4. Volker Dötsch
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  5. Jürgen M. Schmidt
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Correspondence to Frank Löhr or Jürgen M. Schmidt.

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Dedicated to Professor Heinz Rüterjans on the occasion of his 75th birthday.

Electronic supplementary material

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10858_2011_9507_MOESM1_ESM.pdf

Supplementary material includes one Table comparing J coupling constants in protonated and deuterated ubiquitin, one Table of typical multiplet-fit parameters, a Figure demonstrating the weighted superposition of DIPAP constituents, and Figures showing possible origins of valid DIPAP and IPAP multiplet fits that produced discordant coupling constants between both methods, and also a case of heavily overlapping signals. Bruker pulseprograms of the DIPAP sequences of Figure 3 are available on request from author F.L. (PDF 803 kb)

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Löhr, F., Reckel, S., Stefer, S. et al. Improved accuracy in measuring one-bond and two-bond 15N,13Cα coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy. J Biomol NMR 50, 167–190 (2011). https://doi.org/10.1007/s10858-011-9507-3

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