Acid-induced Conformational Changes in Hemagglutinin from Influenza Virus

血凝素（HA）是一种主要的表面膜 负责流感病毒与唾液酸结合的糖蛋白 靶细胞中的含酸受体。融合活性是 触发的pH依赖性构象变化的HA在 内体的酸性环境。HA是一种三聚体蛋白（Mr 195，600），其包含相同亚基的胞外域， 其含有通过连接的两个多肽（HA 1和HA 2）， 二硫键HA的构象稳定性和热稳定性 从流感病毒株X31纯化的流感病毒已经通过以下方法研究： 差示扫描量热法（DSC），圆二色性（CD）， 荧光和超离心。医管局被发现有一个 在pH 7.4至5.4的条件下，具有6个三聚体/玫瑰花结（31 S）的玫瑰花结结构， 含有50 mM磷酸盐-50 mM乙酸盐的混合缓冲液， mM NaCl和1 mM EDTA。低于pH 5.4时，HA制备物是 发现了异质的、不稳定的和完整的流感病毒 布卢门塔尔等人在 分开研究。HA在pH 7.4 ± 1%时的DSC曲线 辛基葡糖苷显示三个结构域，Tm = 66 ± 1 C， 总体[Δ H] = 1000 +- 100 kcal/mol，尽管HA 在洗涤剂存在下解离成三聚体（9.4S）。这 表明，三聚体中的分子间相互作用 玫瑰花结结构对热展开贡献不大 参数随着pH从pH 7.4降低至5.4，Tm 热去折叠的平均值从约66.5 至46.7 ℃和约900至230 kcal/mol。的 酸诱导的不稳定对应于三级结构损失， 如通过近紫外CD和固有的双羟基残基测量的 荧光。有趣的是，温度依赖的远紫外CD 测量结果表明，HA二级结构实际上是 当蛋白质被酸化（pH 7至约90 ℃）时， 5）。质子引起的三级结构的不稳定， 二级结构的明显稳定是 HA.

Hemagglutinin (HA) is a major surface membrane glycoprotein responsible for the binding of influenza virus to sialic acid-containing receptors in target cells. Fusogenic activity is triggered by a pH-dependent conformational change of HA in the acidic milieu of the endosomes. HA is a trimeric protein (Mr 195,600) comprising an ectodomain of identical subunits, each of which contains two polypeptides (HA1 and HA2) linked by a disulfide bond. The conformational and thermal stability of HA purified from influenza strain X31 has been investigated by differential scanning calorimetry (DSC), circular dichroism (CD), fluorescence, and ultracentrifugation. HA was found to have a rosette structure with 6 trimers/rosette (31 S) at pH 7.4 to 5.4 in a mixed buffer containing 50 mM phosphate-50 mM acetate with 100 mM NaCl and 1 mM EDTA. Below pH 5.4, HA preparations were heterogeneous and unstable, and intact influenza virus was found also to be rapidly inactivated at pH < 5.4 by Blumenthal et al. in separate studies. The DSC profiles of HA at pH 7.4 +- 1 % octylglucoside showed three domains with Tm = 66 +- 1 C and overall [Delta H] = 1000 +- 100 kcal/mol even though HA was dissociated to trimers (9.4 S) in the presence of the detergent. This indicates that intermolecular interactions between trimers in the rosette structure contribute little to the thermal unfolding parameters. As the pH was decreased from pH 7.4 to 5.4, the Tm and enthalpic values for thermal unfolding decreased from ca 66.5 to 46.7 deg C and from ca 900 to 230 kcal/mol, respectively. The acid-induced destabilization corresponded to tertiary structure loss, as measured by near UV CD and intrinsic tryptophanyl residue fluorescence. Interestingly, temperature-dependent far UV CD measurements indicated that HA secondary structure was actually stabilized (66 to ca 90 deg C) as the protein was acidified (pH 7 to 5). The proton-induced destabilization of tertiary structure and apparent stabilization of secondary structure are novel features of HA.