SITE-DIRECTED MUTAGENESIS IN HEMOGLOBIN - EFFECT OF SOME MUTATIONS AT PROTEIN INTERFACES

The role of selected amino acid residues in the monomer monomer contacts of Hb A has been studied by site-directed mutagenesis of the alpha chain bearing substitutions in the subunit surfaces. Mutation alpha38Thr-->Trp induced a stabilization of tetrameric Hb-CO with a decrease of the K(d) for the equilibriUM alpha2beta2 double-line arrow pointing left and right 2alphabeta, but had no effect on ligand binding. Mutation alpha40Thr-->Arg resulted in a complete loss of cooperativity in ligand binding. Mutation alpha103His-->Val had no noticeable effect. We also studied the behaviour of isolated, mutated alpha chains with respect to self association: compared to wt alpha chains, mutant alpha38Thr-->Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant alpha103His-->Val showed only a minor stabilization of the alpha2 dimer.