Stereospecific assignment of beta-methylene protons in larger proteins using 3D 15N-separated Hartmann-Hahn and 13C-separated rotating frame Overhauser spectroscopy

J Biomol NMR. 1991 May;1(1):13-22. doi: 10.1007/BF01874566.


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

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Interleukin-1 / chemistry
  • Magnetic Resonance Spectroscopy / methods*
  • Molecular Structure
  • Protein Conformation*
  • Protons
  • Stereoisomerism


  • Amino Acids
  • Interleukin-1
  • Protons