The dimer-tetramer equilibrium of recombinant hemoglobins. Stabilization of the alpha 1 beta 2 interface by the mutation beta(Cys112-->Gly) at the alpha 1 beta 1 interface.
The dimer-tetramer association constants of several recombinant human hemoglobins (in the CO form) have been measured by differential gel filtration. Recombinant human hemoglobin prepared from recombinant beta-chains, and mutant hemoglobins where the substitution was on the surface, beta(Thr4-->Asp), in the heme pocket, beta(Val67-->Thr), at the 2,3-DPG binding site, beta(Val1-->Met+His2del), had a twofold smaller association with respect to natural hemoglobin. In a mutant at the alpha 1 beta 2 interface, beta(Cys93-->Ala), the association constant was decreased three-fold. Conversely, in a mutant at the alpha 1 beta 1 interface, beta(Cys112-->Gly), the association constant was two- and four-fold increased with respect to natural and recombinant human hemoglobin. These differences are energetically very small, consistent with the correct folding of the recombinant hemoglobins. The stabilization of the tetrameric structure by a mutation at the alpha 1 beta 1 interface indicates that structural changes in this interface can be propagated through the protein to the alpha 1 beta 2 interface and, thereby, exert an effect on the allosteric equilibrium.
Fronticelli
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Department of Biochemistry, University of Maryland, School of Medicine, Baltimore 21201.
Gattoni
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Lu
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Brinigar
W S
WS
Bucci
J L
JL
Chiancone
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E
D019794:2,3-Diphosphoglycerate; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002263:Carboxyhemoglobin / Q000737:chemistry*; D002850:Chromatography, Gel; D003545:Cysteine; D004163:Diphosphoglyceric Acids / Q000097:blood; D004355:Drug Stability; D005914:Globins / Q000737:chemistry*; D005998:Glycine; D006418:Heme / Q000378:metabolism; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D014633:Valine
D004163:Diphosphoglyceric Acids; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D019794:2,3-Diphosphoglycerate; D006418:Heme; D005914:Globins; D006441:Hemoglobin A; D002263:Carboxyhemoglobin; D008715:Methionine; D014633:Valine; D003545:Cysteine; D005998:Glycine