Sequence-specific assignments of downfield-shifted amide proton resonances of calmodulin Use of two-dimensional NMR analysis of its tryptic fragments

Abstract

Two-dimensional NMR methods were applied to assign the extremely downfield-shifted amide-proton resonances in the 500-MHz 1H-NMR spectra of the NH 2-terminal fragment of residues 1–75 of calmodulin. The low-field resonances of the 1H-NMR spectra of intact calmodulin were assigned to specific amino acid residues by comparison with spectra of the tryptic fragments of residues 1–75 and 78–148, in both the Ca 2+-free and Ca 2+-bound states. The hydrogen bonding of glycine residues connecting the two amino acid residues at the Z and − Y positions in the octahedral Ca 2+ coordination site was investigated. The Gly 134 in site IV showed a different property from the other glycines, 25, 61 and 98, involved in sites I, II and III, respectively.