Acid-induced Conformational Changes in Hemagglutinin from Influenza Virus

血凝素（HA）是流感病毒与靶细胞中含唾液酸受体结合的主要表面膜糖蛋白。 融合活性由内体酸性环境中HA的pH依赖性构象变化触发。 HA是一种三聚体糖蛋白（约。220，000 MW），其包含相同亚基的胞外域，每个亚基含有通过二硫键连接的两个多肽（HA 1和HA 2）。用差示扫描量热法（DSC）、圆二色性（CD）、荧光、超离心和电子显微镜研究了从流感病毒株X31中纯化的HA的构象和热稳定性。 已发现HA在含有50 mM磷酸盐-50 mM乙酸盐与100 mM NaCl和1 mM EDTA的混合缓冲液中在pH 7.4至5.4下具有玫瑰花结结构，其中5-6个三聚体/玫瑰花结（31-35 S）。 低于pH5.4时，HA制剂是异质的和不稳定的，并且布卢门塔尔等人发现完整的流感病毒也在低于pH5.4时快速失活。在不存在和存在1%辛基葡糖苷的情况下，在pH7.4时HA的DSC曲线的分析显示三个结构域，分别具有Tm = 66和65 ℃，和900 kcal/mol三聚体的总解折叠焓，即使HA在去污剂存在下解离成三聚体（9.4 S）。 在这两种情况下，合作比CR（量热范特霍夫焓）为3.0，表明HA亚基在三聚体中独立展开。 此外，在玫瑰花结结构中的三聚体之间的分子间相互作用似乎贡献不大的热展开参数。 随着pH从pH7.4降低到5.4（样品在37 ℃下预孵育30分钟），转变温度（Tm）和解折叠温度分别从66 ℃降低到47 ℃和从900 kcal/mol三聚体降低到240 kcal/mol三聚体，相应的CR从3.0降低到1.2. 酸诱导的不稳定对应于三级结构的损失，如通过近UV CD和固有的Dahanyl残基荧光所测量的。 有趣的是，温度依赖性远紫外CD测量表明，HA二级结构实际上是稳定的（从66至> 90 ℃），因为蛋白质被酸化（pH 7至5）。 质子诱导的三级结构的不稳定性和二级结构的表观稳定性是HA的新特征。光谱和DSC实验已经在今年进行，以重新确定与新鲜HA制剂的热去折叠HA的一些参数。 此外，在pH 7.4的HA的部分比体积已被确定为0.73毫升/克的同时沉降平衡的HA roximine在水溶液和氧化氘缓冲液。

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 glycoprotein (ca. 220,000 MW) 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, ultracentrifugation, and electron microscopy. HA has been found to have a rosette structure with 5-6 trimers/rosette (31-35 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 are heterogeneous and unstable, and intact influenza virus has been found also to be rapidly inactivated at below pH 5.4 by Blumenthal et al. Analyses of DSC profiles of HA at pH 7.4 in the absence and presence of 1% octylglucoside show three domains with Tm = 66 and 65 C, respectively, and an overall unfolding enthalpy of 900 kcal/mol of trimer even though HA is dissociated to trimers (9.4 S) in the presence of the detergent. In both cases, the cooperative ratio CR (calorimetric to van't Hoff enthalpy) is 3.0, suggesting that HA subunits unfold independently in the trimer. Moreover, intermolecular interactions between trimers in the rosette structure appears to contribute little to the thermal unfolding parameters. As the pH is decreased from pH 7.4 to 5.4 (with a 30 min preincubation of samples at 37 C), both transition temperatures (Tm) and unfolding enthalpies decrease from 66 to 47 C and from 900 to 240 kcal/mol trimer, respectively, with corresponding decreases in CR from 3.0 to 1.2. The acid-induced destabilization corresponds 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 is actually stabilized (from 66 to > 90 C) as the protein is acidified (pH 7 to 5). The proton-induced destabilization of tertiary structure and apparent stabilization of secondary structure are novel features of HA.Spectral and DSC experiments have been performed this year to redetermine some of the parameters for thermal unfolding HA with fresh preparations of HA. Also, the partial specific volume of HA at pH 7.4 has been determined to be 0.73 ml/g by simultaneous sedimentation equilibrium of HA rosettes in aqueous and deuterium oxide buffer.