Resonance assignments in the proton-NMR spectrum of carbonmonoxy hemoglobin by two-dimensional methods.

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (M_r = 64 500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heine substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in β subunits which, in solution, appears to have stronger contacts with the heine groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (M_r ≤ 65 000). [ABSTRACT FROM AUTHOR]