NMR Investigation of the Dynamics of Tryptophan Side-chains in Hemoglobins

Abstract

NMR relaxation measurements of 15N spin–lattice relaxation rate ( R 1), spin–spin relaxation rate ( R 2), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7 T and 14.1 T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or ( 2H, 15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (βD99N), rHb (αY42D/βD99N), and rHb (αV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (α14, β15, and β37) in Hb A for each αβ dimer. These Trp residues are located in important regions of the Hb molecule, i.e. α14Trp and β15Trp are located in the α 1β 1 subunit interface and β37Trp is located in the α 1β 2 subunit interface. The relaxation experiments show that amino acid substitutions in the α 1β 2 subunit interface can alter the dynamics of β37Trp. The transverse relaxation rate ( R 2) for β37Trp can serve as a marker for the dynamics of the α 1β 2 subunit interface. The relaxation parameters of deoxy-rHb Kemspey (βD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (αY42D/βD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY–CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of β37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure–function relationship in hemoglobin.