ACID-INDUCED CONFORMATIONAL CHANGES IN HEMAGGLUTININ FROM INFLUENZA VIRUS

血凝素（HA）是一种主要的表面膜糖蛋白，负责将流感病毒与靶细胞中含有唾液酸的受体结合。核内体酸性环境中HA的ph依赖性构象变化触发了促聚变活性。透明质酸是一种三聚体糖蛋白（约220,000 MW），由相同亚基的外结构域组成，每个外结构域包含两个由二硫键连接的多肽（HA1和HA2）。用差示扫描量热法（DSC）、圆二色法（CD）、荧光法和超离心法研究了从流感病毒株X31中纯化的透明质酸的构象和热稳定性。在pH 7.4 ~ 5.4的混合缓冲液中，发现HA具有6个三聚体/莲座结构（约33 S），其中含有50 mM磷酸盐-50 mM醋酸盐，100 mM NaCl和1 mM EDTA。Blumenthal等人发现，在pH低于5.4时，HA制剂是异质且不稳定的，完整的流感病毒在pH低于5.4时也能迅速灭活。在pH为7.4时，在不含和存在1%辛基葡萄糖苷的情况下，对HA的DSC谱进行分析，结果显示，即使HA在洗涤剂的存在下解离成三聚体（9.4 S）， Tm = 66 +/- 1 C和总体[δ H] = 1000 +/- 100 kcal/mol的三个结构域。这表明玫瑰结构中三聚体之间的分子间相互作用对热展开参数的影响很小。当pH值从pH 7.4降低到pH 5.4时，热展开的Tm和焓值分别从66.5℃和900 kcal/mol降低到230 kcal/mol。通过近紫外CD和固有色氨酸残基荧光测量，酸诱导的不稳定与三级结构损失相对应。有趣的是，依赖于温度的远紫外CD测量表明，当蛋白质酸化（pH 7至5）时，HA二级结构实际上是稳定的（从66至约90℃）。质子诱导三级结构的失稳和二级结构的明显稳定是透明质酸的新特征。正在进行最后的光谱和DSC实验，以用新的HA制剂重新确定上述一些值，以及通过沉淀平衡确定部分比容。-血凝素、流感病毒、寡聚物结构、热展开、量热法、圆二色性

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) comprised of 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 (ca. 33 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 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 percent 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 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 (from 66 to ca. 90 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. Final spectral and DSC experiments are being performed to redetermine some of the above values with a fresh preparation of HA, as well as a determination of the partial specific volume by sedimentation equilibrium. - Hemagglutinin, influenza virus, oligomeric structure, thermal unfolding, calorimetry, circular dichroism