Paramagnetic Ion Induced Perturbations in the ¹H NMR Spectrum of Lysozyme: A Reassignment of the Tryptophan Indole NH Resonances

In H₂O the 360-MH_z ¹H NMR spectrum of the egg white lysozyme, an enzyme which has six tryptophan residues, contains only five resolvable indole N H resonances. These peaks have been assigned to specific tryptophan residues on the basis of the shift perturbations induced by Co²⁺ and line broadenings induced by Gd³⁺. Since each of these perturbations obeys a different geometric relationship, the agreement of the two sets of assignments provides a check of the overall method. The results of inhibitor binding studies, chemical modification experiments, and deuterium isotope exchange rates are discussed in terms of the new assignments. Apart from any specific conclusions reached on HEW lysozyme it is clear that for many macromolecules the line broadenings induced by Gd³⁺ can be analyzed in terms of absolute metal proton distances. In making these assignments we have made use of the crystallographic data for HEW lysozyme. In general, the application of the methodology presented here is restricted to those biomolecules whose structure has been determined by an independent method.