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 alpha 38Thr-->Trp induced a stabilization of tetrameric Hb-CO with a decrease of the Kd for the equilibrium alpha 2 beta 2<==>2 alpha beta, but had not effect on ligand binding. Mutation alpha 40Thr-->Arg resulted in a complete loss of cooperativity in ligand binding. Mutation alpha 103His-->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 alpha 38Thr-->Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant alpha 103His-->Val showed only a minor stabilization of the alpha 2 dimer.
Department of Biochemical Sciences, University of Rome La Sapienza, Italy.
B
B
Vallone
P
P
Vecchini
V
V
Cavalli
M
M
Brunori
D000494:Allosteric Regulation; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D002248:Carbon Monoxide / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014633:Valine / Q000737:chemistry / Q000235:genetics
D008024:Ligands; D013912:Threonine; D006639:Histidine; D002248:Carbon Monoxide; D014364:Tryptophan; D006441:Hemoglobin A; D001120:Arginine; D014633:Valine; D010100:Oxygen