High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization.
The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.
Craescu
C T
CT
Schaeffer
C
C
Mispelter
J
J
Garin
J
J
Rosa
J
J
D004563:Electrochemistry; D014644:Genetic Variation; D006441:Hemoglobin A; D006455:Hemoglobins, Abnormal; D006639:Histidine; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010108:Oxyhemoglobins; D011487:Protein Conformation
D006455:Hemoglobins, Abnormal; D010108:Oxyhemoglobins; D006639:Histidine; D006441:Hemoglobin A