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
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Linear Mode
