Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains
Spectroscopic and calorimetric analyses of invasion plasmid antigen D (IpaD) from Shigella flexneri reveal the presence of two structural domains
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DOI：
10.1021/bi060625v
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发表时间：
2006-08-01
期刊：
影响因子：
2.9
通讯作者：
Picking, Wendy L.
中科院分区：
文献类型：
--
作者：
Espina, Marianela;Ausar, S. Fernando;Picking, Wendy L.
福氏志贺氏菌是一种兼性细胞内病原体，可引起人类严重胃肠炎。入侵质粒抗原 D (IpaD) 是志贺氏菌入侵肠道细胞的重要参与者，但没有详细的结构信息来帮助了解 IpaD 在入侵中的作用或其与其他入侵蛋白的相互作用。因此，使用傅里叶变换红外 (FTIR)、圆二色性 (CD) 以及内在和外在荧光光谱研究了 IpaD 以及选定的 IpaD 缺失突变体的二级和三级结构和热稳定性。热展开的能量学也通过差示扫描量热法（DSC）进行了评估。通过 CD 和 FTIR 进行的二级结构分析表明，IpaD 主要是 R 螺旋，具有分子内卷曲线圈的特征。热研究表明，IpaD 的展开是一个复杂的过程，由以 59 和 80 摄氏度附近为中心的两个转变组成。对完整蛋白质和选定的缺失突变体获得的数据进行比较表明，较低的温度转变是一个可逆事件，可归因于位于 IpaD N 末端的小结构域的展开。相比之下，所提出的主要且高度稳定的C端结构域的热解折叠是不可逆的并导致蛋白质聚集。当结果综合起来时，它们强烈支持 IpaD 具有两个独立折叠域的观点。
Shigella flexneri is a facultative intracellular pathogen that causes severe gastroenteritis in humans. Invasion plasmid antigen D ( IpaD) is an essential participant in Shigella invasion of intestinal cells, but no detailed structural information is available to help understand the proposed role of IpaD in invasion or its interaction with other invasion proteins. Therefore, the secondary and tertiary structure and thermal stability of IpaD as well as selected IpaD deletion mutants were investigated using Fourier transform infrared ( FTIR), circular dichroism ( CD), and both intrinsic and extrinsic fluorescence spectroscopies. The energetics of thermal unfolding were also evaluated by differential scanning calorimetry (DSC). Secondary-structure analysis by CD and FTIR suggests that that IpaD is primarily R-helical with characteristics of a intramolecular coiled coil. Thermal studies revealed that the unfolding of IpaD is a complex process consisting of two transitions centered near 59 and 80 degrees C. A comparison of the data obtained with the intact protein and selected deletion mutants indicated that the lower temperature transition is a reversible event attributable to the unfolding of a small domain located at the N terminus of IpaD. In contrast, the thermal unfolding of the proposed major and highly stable C-terminal domain was irreversible and led to protein aggregation. When the results are taken together, they strongly support the idea that IpaD has two independent folding domains.