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），由相同亚基的胞外域组成，每个亚基含有两个通过二硫键连接的多肽（HA 1和HA 2）。用差示扫描量热法（DSC）、圆二色性（CD）、荧光和超离心法研究了从流感病毒株X31中纯化的HA的构象和热稳定性。发现HA具有6个三聚体/玫瑰花结的玫瑰花结结构（约100 μ g）。33 S）在pH 7.4至5.4下在含有50 mM磷酸盐-50 mM乙酸盐与100 mM NaCl和1 mM EDTA的混合缓冲液中进行。低于pH 5.4时，HA制剂是不均匀和不稳定的，布卢门塔尔等人发现，完整的流感病毒在低于pH5.4时也能迅速灭活。在pH7.4时，在不存在和存在1%辛基葡糖苷的情况下，HA的DSC图谱分析显示了三个结构域，Tm = 66 +/- 1 ℃，总[Δ H] = 1000 +/- 100 kcal/mol，即使HA在去污剂的存在下解离成三聚体（9.4S）。这表明玫瑰花结结构中三聚体之间的分子间相互作用对热去折叠参数的贡献很小。随着pH从pH 7.4降低到5.4，热去折叠的Tm和热变性值从约0.05降低到约0.05，热去折叠的Tm和热变性值从约0.05降低到约0.05。66.5到46.7 C和从约. 900至230千卡/摩尔。酸诱导的不稳定对应于三级结构的损失，如通过近紫外CD和固有的Dahanyl残基荧光测量的。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到约100 μ m）。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和二级结构的表观稳定是HA的新特征。正在进行最终的光谱和DSC实验，以重新确定一些上述值与新鲜制备的HA，以及通过沉降平衡的部分比容的测定。- 血凝素，流感病毒，寡聚体结构，热去折叠，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