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Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

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Summary

3J xβ coupling constants and complementary nuclear Overhauser data on the intraresidue CxH−CβH distances form an essential part of the data needed to obtain stereospecific assignments of β-methylene protons in proteins. In this paper we show that information regarding the magnitude of the3J xβ coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D15N-separated1H−1H Hartmann-Hahn1H−15N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue CxH−CβH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D13C-separated1H−1H rotating frame Overhauser effect1H−13C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1β, a protein of 153 residues and 17.4 kDa molecular weight.

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Abbreviations

1L-1β:

interleukin-1β

NOE:

nuclear Overhauser effect

ROE:

rotating frame Overhauser effect

HOHAHA:

homonuclear Hartmann-Hahn spectroscopy

NOESY:

nuclear Overhauser enhancement spectroscopy

ROESY:

rotating frame Overhauser spectroscopy

HMQC:

heteronuclear multiple quantum coherence spectroscopy

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Authors and Affiliations

  1. Laboratory of Chemical Physics, Building 2, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892, Bethesda, MD, USA

    G. Marius Clore, Ad Bax & Angela M. Gronenborn

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  1. G. Marius Clore
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  2. Ad Bax
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  3. Angela M. Gronenborn
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Clore, G.M., Bax, A. & Gronenborn, A.M. Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy. J Biomol NMR 1, 13–22 (1991). https://doi.org/10.1007/BF01874566

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  • DOI: https://doi.org/10.1007/BF01874566

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