Identification using 1H NMR spectroscopy of slowly exchanging amide hydrogens of hen lysozyme in solution

Abstract

Resonances of over 20 of the most slowly exchanging amide hydrogens have been identified and assigned in the ¹H NMR spectrum of hen lysozyme. This was achieved by combining information about spin-spin coupling patterns with nuclear Overhauser enhancement measurements. A computer-based search program was used to permit the assumptions and constraints in this procedure to be closely defined and to reveal possible ambiguities. In addition, experimental values of coupling constants were compared with values calculated on the basis of the torsion angles found in the crystal structure. The close correlation between these gave further confidence in the assignment procedures and provided information concerning the nature of fluctuations about the average solution structure. The very slowly exchanging amide hydrogens are largely buried in α-helical and β-sheet regions of the protein structure.