Baudin-Chich V, Pagnier J, Marden M, Bohn B, Lacaze N, Kister J, Schaad O, Edelstein S J, Poyart C
Unité 299 Institut National de la Santé et de la Recherche Médicale, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Proc Natl Acad Sci U S A. 1990 Mar;87(5):1845-9. doi: 10.1073/pnas.87.5.1845.
Polymerization of the deoxy form of sickle cell hemoglobin (Hb S; beta 6 Glu----Val) involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the beta 6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. This interaction constitutes the major lateral contact between the two strands in a pair. To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a polymerizing Hb with isoleucine at the beta 6 position (beta E6I) by site-directed mutagenesis. The mutated beta chains were produced in Escherichia coli and reassembled into functional tetramers with native alpha chains. Compared to native Hb S, the beta E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth. The solubility of the beta E6I mutant Hb is half that of native Hb S. Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the beta 6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.
镰状细胞血红蛋白（Hb S；β6 谷氨酸→缬氨酸）的脱氧形式的聚合涉及疏水和静电分子间接触。这些相互作用驱使突变分子形成由七对链组成的长纤维棒。Hb S的X射线晶体学和纤维的电子显微镜图像重建揭示了每个分子的一个β6缬氨酸（供体位点）与相邻四聚体EF角处的受体位点之间显著的互补性。这种相互作用构成了一对中两条链之间的主要横向接触。为了评估这种关键疏水接触在聚合物形成中的相对重要性，我们通过定点诱变在β6位置产生了异亮氨酸的聚合Hb（βE6I）。突变的β链在大肠杆菌中产生，并与天然α链重新组装成功能性四聚体。与天然Hb S相比，βE6I突变体在1.8 M磷酸盐缓冲液中聚合更快且延迟时间缩短，表明纤维生长前核的稳定性增加。βE6I突变体Hb的溶解度是天然Hb S的一半。供体-受体相互作用的计算机模拟表明，供体位点异亮氨酸侧链的存在导致与受体位点的接触增加和埋藏表面积增加，这与异亮氨酸残基更高的疏水性一致。预测的和实验观察到的溶解度差异之间的一致性表明，β6缬氨酸或异亮氨酸侧链从水转移到疏水环境足以解释这些观察结果。
