Roles of Fe-Histidine bonds in stability of hemoglobin: recognition of protein flexibility by Q Sepharose
Roles of Fe-Histidine bonds in stability of hemoglobin: recognition of protein flexibility by Q Sepharose
复制标题
Fe-组氨酸键在血红蛋白稳定性中的作用：Q Sepharose 识别蛋白质柔性
DOI：
10.1016/j.bpj.2021.05.014
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发表时间：
2021
影响因子：
3.4
通讯作者：
Masako Nagai
中科院分区：
文献类型：
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作者：
Shigenori Nagatomo;Teizo Kitagawa;Masako Nagai
使用各种突变体，我们研究了 Fe-组氨酸 (F8) 键在人血红蛋白 (Hb) 协同 O2 结合中的作用以及 α2β2 四聚体中 α 和 β 亚基之间的作用差异。血红素腔内发生突变的 Hb 变体表现出意想不到的特征。当β突变体rHb（βH92G）的β亚基的近端组氨酸（His F8）被甘氨酸（Gly）取代时，经过离子交换色谱（Q Sepharose柱）并在咪唑存在下用NaCl浓度梯度洗脱，产生两个大峰，而相应的α突变体rHb（αH87G）则产生与Hb A相似的单峰。等电聚焦电泳显示各峰下的β突变型rHb蛋白具有相同的等电点。通过 Sephadex G-75 凝胶过滤、远紫外 CD、1H NMR 和共振拉曼光谱进一步表征每个峰下的蛋白质。我们发现rHb（βH92G）以αβ二聚体和α2β2四聚体的混合物形式存在，并且血红素从二聚体的一部分中的β亚基中释放出来。根据拉曼相对强度，释放的血红素的大约量估计高达 30%。需要强调的是，Q Sepharose 柱可以通过改变多孔珠的排出速率来区分具有相同等电点的蛋白质的结构灵活性的差异。因此，Barrick 等人首先描述了 Fe-His (F8) 键在稳定 Hb 四聚体中的作用。在这项研究中得到了证实。此外，本研究发现，β-亚基中的特定 Fe-His 键可最大限度地降低球蛋白结构的灵活性。
Using various mutants, we investigated to date the roles of the Fe-histidine (F8) bonds in cooperative O2binding of human hemoglobin (Hb) and differences in roles betweenα-andβ-subunits in theα2β2tetramer. An Hb variant with a mutation in the heme cavity exhibited an unexpected feature. When theβmutant rHb (βH92G), in which the proximal histidine (His F8) of theβ-subunit is replaced by glycine (Gly), was subjected to ion-exchange chromatography (Q Sepharose column) and eluted with an NaCl concentration gradient in the presence of imidazole, yielded two large peaks, whereas the correspondingα-mutant, rHb (αH87G), gave a single peak similar to Hb A. Theβ-mutant rHb proteins under each peak had identical isoelectric points according to isoelectric focusing electrophoresis. Proteins under each peak were further characterized by Sephadex G-75 gel filtration, far-UV CD,1H NMR, and resonance Raman spectroscopy. We found that rHb (βH92G) exists as a mixture ofαβ-dimers andα2β2tetramers, and that hemes are released fromβ-subunits in a fraction of the dimers. An approximate amount of released hemes were estimated to be as large as 30% with Raman relative intensities. It is stressed that Q Sepharose columns can distinguish differences in structural flexibility of proteins having identical isoelectric points by altering the exit rates from the porous beads. Thus, the role of Fe-His (F8) bonds in stabilizing the Hb tetramer first described by Barrick et al. was confirmed in this study. In addition, it was found in this study that a specific Fe-His bond in theβ-subunit minimizes globin structural flexibility.