Acid-induced Conformational Changes in Hemagglutinin from Influenza Virus

血凝素(HA)是一种主要的表面膜 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 由羟基磷灰石的pH依赖构象变化触发 内小体的酸性环境。HA是一种三聚体蛋白质(Mr 195,600)包括相同亚基的胞外域，每个亚基 它包含两个多肽(HA1和HA2)，通过一个 二硫键。羟基磷灰石的构象和热稳定性 从流感病毒X31株中提纯的病毒已被 差示扫描量热法(DSC)、圆二色谱(CD)、 荧光和超速离心法。HA被发现有一种 含6个三聚体的玫瑰花环结构(31 S)，pH 7.4~5.4 含有50毫米磷酸盐-50毫米醋酸盐的混合缓冲液 MM氯化钠和1 mM EDTA。在pH 5.4以下，透明质酸制剂 异质性和不稳定性，完整的流感病毒被发现 也被Blumenthal等人在pH&lt；5.4下快速灭活。在……里面 单独的研究。PH 7.4+-1%时透明质酸的DSC图谱 辛基葡萄糖苷显示三个结构域，Tm=66+1C和 总体而言，[Delta H]=1000+-100千卡/摩尔，即使HA 在洗涤剂存在下解离成三聚体(9.4 S)。这 表明三聚体之间的分子间相互作用 玫瑰花环结构对热展开的贡献很小 参数。当pH从7.4时降至5.4时，TM 热展开的热值从约66.5下降。 分别为46.7℃和900~230千卡/摩尔。这个 酸诱导的失稳对应于三级结构的损失， 用近紫外CD和本征色氨酸残基测定 荧光。有趣的是，依赖温度的远紫外光CD 测量表明，HA的二级结构实际上是 当蛋白质酸化(pH 7至90℃)时稳定(66℃至90℃) 5)。质子引起的三级结构失稳和 表面上稳定的二级结构是新的特征 哈哈。

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.