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

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


Abstract An extension to HN(CO-α/β-N,Cα-J)-TROSY (Permi and Annila in J Biomol NMR 16:221â??227, 2000) is proposed that permits the simultaneous determination of the four coupling constants 1 J Nâ?²(i)Cα(i), 2 J HN(i)Cα(i), 2 J Cα(iâ??1)Nâ?²(i), and 3 J Cα(iâ??1)HN(i) in 15N,13C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the 2 J CαNâ?² 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 CαNâ?² and 1 J Nâ?²Cα (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.

• Content Type Journal Article
• Pages 1-24
• DOI 10.1007/s10858-011-9507-3
• Authors
  • Frank Löhr, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
  • Sina Reckel, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
  • Susanne Stefer, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
  • Volker Dötsch, Institute of Biophysical Chemistry, Center for Biomolecular Magnetic Resonance, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
  • Jürgen M. Schmidt, School of Biosciences, University of Kent, Giles Lane, Canterbury, Kent, CT2 7NJ UK

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738

Source: Journal of Biomolecular NMR