Kiger L, Klinger A L, Kwiatkowski L D, De Young A, Doyle M L, Holt J M, Noble R W, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Mar 31;37(13):4336-45. doi: 10.1021/bi970868a.
In human hemoglobin (Hb) the beta37 tryptophan residue (betaW37), located at the hinge region of the alpha1beta2 interface, forms many contacts with alpha subunit residues of the opposite dimer, in both the T and R quaternary structures. We have carried out equilibrium O2 binding studies on a series of recombinant Hbs that have mutations at this residue site: betaW37Y, betaW37A, betaW37G, and betaW37E. Binding isotherms measured at high concentrations of these mutants were found to be shifted toward increased affinity and decreased cooperativity from that of the normal HbA0 tetramer. Analysis of these binding isotherms indicated that amino acid substitutions at the beta37 position could both destabilize the tetrameric form of the mutants relative to their constituent dimers and also alter cooperativity of the intact tetrameric species. These alterations from wild-type function are dependent on the particular side chain substituted, with the magnitude of change increasing as Trp is substituted by Tyr, Ala, Gly, and Glu. The dimer to tetramer assembly free energy of deoxy-betaW37E, the most perturbed mutant in the series, was measured using analytical gel chromatography to be 9 kcal/tetramer less favorable than that of deoxy HbA0. Stabilizing the betaW37E tetramer by addition of IHP, or by cross-linking at the alphaK99 positions, does not restore normal O2 binding behavior. Thermodynamic parameters of all the mutants were found to correlate with their CO binding rates and with their high-resolution X-ray crystal structures (see accompanying papers: Kwiatkowski et al. (1998) Biochemistry 37, 4325-4335; Peterson & Friedman (1998) Biochemistry 37, 4346-4357; Kavanaugh et al. (1998) Biochemistry 37, 4358-4373].
在人血红蛋白（Hb）中，位于α1β2界面铰链区的β37色氨酸残基（βW37），在T和R四级结构中均与相对二聚体的α亚基残基形成许多接触。我们对一系列在该残基位点发生突变的重组Hb进行了平衡O2结合研究：βW37Y、βW37A、βW37G和βW37E。发现在高浓度下测量的这些突变体的结合等温线相对于正常HbA0四聚体向亲和力增加和协同性降低的方向移动。对这些结合等温线的分析表明，β37位置的氨基酸取代既可能使突变体的四聚体形式相对于其组成二聚体不稳定，也会改变完整四聚体物种的协同性。与野生型功能的这些改变取决于所取代的特定侧链，随着色氨酸被酪氨酸、丙氨酸、甘氨酸和谷氨酸取代，变化程度增加。使用分析凝胶色谱法测得该系列中受干扰最大的突变体脱氧-βW37E的二聚体到四聚体组装自由能比脱氧HbA0的不利9千卡/四聚体。通过添加IHP或在αK99位置交联来稳定βW37E四聚体，并不能恢复正常的O2结合行为。发现所有突变体的热力学参数与其CO结合速率及其高分辨率X射线晶体结构相关（见随附论文：Kwiatkowski等人（1998年）《生物化学》37卷，4325 - 4335页；Peterson和Friedman（1998年）《生物化学》37卷，4346 - 4357页；Kavanaugh等人（1998年）《生物化学》37卷，4358 - 4373页）。
