EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein
EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein
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DOI：
10.1111/j.1742-4658.2004.04513.x
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发表时间：
2005-02-01
期刊：
影响因子：
5.4
通讯作者：
Yanagihara, I
中科院分区：
文献类型：
--
作者：
Hamada, D;Kato, T;Yanagihara, I
EspB，大肠杆菌O 157 III型分泌系统的毒力因子的结构特性进行了表征。远紫外和近紫外CD光谱，记录pH值1.0和pH值7.0之间，表明该蛋白质假定α-螺旋结构，并可能存在一些酪氨酸三级接触。所有酪氨酸侧链暴露于水，如通过丙烯酰胺荧光猝灭光谱法测定的。在EspB的存在下，在pH 2.0下观察到8-苯胺基萘-1-磺酸盐的荧光强度增加，而在pH 7.0下没有观察到荧光的这种增加。这些数据表明在pH 2.0下形成熔融球状态。在低pH值的EspB的不稳定显示尿素展开转换，监测远紫外CD光谱。沉降平衡研究的结果表明，EspB在pH 7.0时呈现单体形式，尽管其斯托克斯半径（通过多角度激光散射估计）是预期EspB单体球形结构的两倍。这些数据表明，EspB，在pH 7.0，假设一个相对扩展的构象。EspB N-15-H-1杂原子单量子相关光谱在pH 2.0和7.0时的化学位移模式与尿素解折叠的EspB的化学位移模式定性相似。总之，这里报道的EspB的性质提供了证据表明EspB是天然部分折叠的蛋白质，但具有比传统熔融球更少的暴露的疏水表面。当EspB在宿主细胞的细菌感染期间与各种生物分子相互作用时，EspB的这种结构特征可能是有利的。
The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB N-15-H-1 heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.