Molecular engineering of a polymer of tetrameric hemoglobins.
Molecular engineering of a polymer of tetrameric hemoglobins.
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四聚血红蛋白聚合物的分子工程。
DOI：
10.1002/prot.1086
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Vasquez,GB
中科院分区：
文献类型：
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作者：
Fronticelli,C;Arosio,D;Bobofchak,KM;Vasquez,GB
我们设计了一种重组突变型人血红蛋白，Hb Prisca β（S9 C + C93 A + C112 G），它以聚合形式组装。在Hb波尔图阿莱格雷（β 9 Ser → Cys）的模型上，通过在β9位引入的半胱氨酸残基之间形成分子间S-S键来获得聚合（Bonaventura和Riggs，Science 1967;155：800-802）。Cβ93和Cβ112被替换，以防止在表达、组装和聚合过程中形成假S-S键。动态光散射测量表明，最终聚合产物主要由6至8个四聚体血红蛋白分子形成。样品多分散性Q = 0.07 ± 0.02，与纯化人血红蛋白的多分散性Q= 0.02 ± 0.02相似，与良好的均匀度一致。在强还原剂的存在下，聚合物恢复到其四聚体形式。在解聚过程中，观察到的流体动力学半径和系统的光散射，这反过来又是成比例的蛋白质的质量之间的直接相关性。我们认为这表明血红蛋白分子紧密地堆积在聚合物中，没有空隙。血红蛋白分子的紧密堆积表明聚合物具有球形形状，因此可以估计其半径。一个有限数量的四聚血红蛋白分子的安排的说明。这种聚合物的构象和功能特性，如血红素口袋构象，变性稳定性，自氧化速率，氧亲和力和协同性，保持类似的四聚体人血红蛋白。蛋白质2001;44：212-222.© 2001 Wiley利斯公司
We have engineered a recombinant mutant human hemoglobin, Hb Prisca β(S9C+C93A+C112G), which assembles in a polymeric form. The polymerization is obtained through the formation of intermolecular SS bonds between cysteine residues introduced at position β9, on the model of Hb Porto Alegre (β9Ser → Cys) (Bonaventura and Riggs, Science 1967;155:800–802). Cβ93 and Cβ112 were replaced in order to prevent formation of spurious SS bonds during the expression, assembly, and polymerization events. Dynamic light scattering measurements indicate that the final polymerization product is mainly formed by 6 to 8 tetrameric hemoglobin molecules. The sample polydispersityQ= 0.07 ± 0.02, is similar to that of purified human hemoglobin (Q= 0.02 ± 0.02), consistent with a good degree of homogeneity. In the presence of strong reducing agents, the polymer reverts to its tetrameric form. During the depolymerization process, a direct correlation is observed between the hydrodynamic radius and the light scattering of the system, which, in turn, is proportional to the mass of the protein. We interpret this to indicate that the hemoglobin molecules are tightly packed in the polymer with no empty spaces. The tight packing of the hemoglobin molecules suggests that the polymer has a globular shape and, thus, allows estimation of its radius. An illustration of an arrangement of a finite number of tetrameric hemoglobin molecules is presented. The conformational and functional characteristics of this polymer, such as heme pocket conformation, stability to denaturation, autoxidation rate, oxygen affinity, and cooperativity, remain similar to those of tetrameric human hemoglobin. Proteins 2001;44:212–222. © 2001 Wiley‐Liss, Inc.