Mechanism of Human Apohemoglobin Unfolding.
Mechanism of Human Apohemoglobin Unfolding.
复制标题
人类脱辅血红蛋白的展开机制。
DOI：
10.1021/acs.biochem.6b01235
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Olson,JohnS
中科院分区：
文献类型：
--
作者：
Samuel,PremilaP;Ou,WilliamC;PhillipsJr,GeorgeN;Olson,JohnS
从人血红蛋白（Hb）中去除血红素导致脱辅基珠蛋白异二聚体的形成。用盐酸胍（GdnHCl）滴定该脱辅基二聚体导致指示两个不同步骤的双相展开曲线。最初，血红素口袋展开并产生二聚体中间体，其中原始螺旋度损失约50%，但α1β 1界面仍然完整。在较高的盐酸钆浓度下，该中间体解离成未折叠的单体。通过比较成年人血红蛋白A（HbA）、重组胎儿人血红蛋白（HbF）、α链间单甘氨酸连接体交联的重组血红蛋白以及具有显著稳定两种亚基中天然构象的非极性血红素口袋突变的重组血红蛋白的GdnHCl滴定，验证了该结构解释。第一阶段的apoHb展开是独立的蛋白质浓度，遗传交联的影响不大，但显着转向更高的盐酸钆浓度的稳定远端口袋突变。第二阶段取决于蛋白质浓度，并通过遗传交联转移到更高的GdnHCl浓度。这个apoHb解折叠模型使我们能够定量apoHbA和apoHbF之间稳定性的细微差异，这表明β和γ血红素口袋具有相似的稳定性，而α1γ 1界面比α1β 1界面更耐解离。
Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1interface is more resistant to dissociation than the α1β1interface.