Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotas ostreatus
Effect of pH on the pore forming activity and conformational stability of ostreolysin, a lipid raft-binding protein from the edible mushroom Pleurotas ostreatus
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DOI：
10.1021/bi051013y
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发表时间：
2005-08-23
期刊：
影响因子：
2.9
通讯作者：
Ulrih, NP
中科院分区：
文献类型：
--
作者：
Berne, S;Sepcic, K;Ulrih, NP
平菇溶菌素是平菇(Pleurotus Ostreatus)中的一种致孔蛋白，属于大豆溶素蛋白家族，是细菌、霉菌、蘑菇和植物中发现的一组新的小酸性蛋白质。它与脂筏结合，并与富含胆固醇的脂类结构域特异性地相互作用。在本研究中，根据测序结果，Ostreolysin被归类为单域All-beta结构蛋白。通过CD、UV吸收和本征色氨酸荧光等方法研究了pH诱导和热诱导下Ostreolysin的去折叠，以表征与其功能性质相关的构象转变，即与脂膜结合、脂泡上的造孔活性和溶血。在25℃和pH 6~9之间，ostreolysin采用热力学稳定的单体类天然构象，具有刚性的三级结构和以β-折叠为主的二级结构。在pH 2和3之间，蛋白质经历了一个不可逆的转变，转变为结合ANS的部分未折叠的熔融球状状态，并显示出破坏的三级结构和增强的非天然α-螺旋结构。功能研究表明，与Colicins和其他一些细菌致孔毒素不同，酸诱导的Ostreolysin熔融球状状态与脂质结合和毛孔形成无关。相反，致密的天然状态是与胆固醇/鞘磷脂多层囊泡结合所必需的，最适pH范围为6至7，并且对于孔形成和溶血来说，最大限度地在pH 7至8之间。
Ostreolysin, a pore-forming protein from the edible oyster mushroom (Pleurotus ostreatus), is a member of the aegerolysin protein family, a novel group of small acidic proteins found in bacteria, molds, mushrooms, and plants. It binds to lipid rafts and interacts specifically with cholesterol-rich lipid domains. In this study, ostreolysin was classified as a single-domain all beta-structured protein on the basis of cDNA sequencing. pH-induced and thermally induced unfolding of ostreolysin was studied by means of CD, UV absorption, and intrinsic tryptophan fluorescence to characterize conformational transitions associated with its functional properties, i.e., binding to lipid membranes, pore forming activity on lipid vesicles, and hemolysis. At 25 degrees C and between pH 6 and 9, ostreolysin adopted a monomeric and thermodynamically stable nativelike conformation, characterized by rigid tertiary structure and predominantly beta-sheet secondary structure. Between pH 2 and 3, the protein underwent an irreversible transition to a partially unfolded, molten globule-like state which bound ANS, and exhibited disrupted tertiary structure and enhanced non-native alpha-helical structure. Functional studies showed that, unlike colicins and some other bacterial pore-forming toxins, the acid-induced molten globule-like state of ostreolysin is not relevant for lipid binding and pore formation. Instead, the compact native state was necessary for binding to cholesterol/sphingomyelin multilamellar vesicles, optimally in the pH range from 6 to 7, and for pore formation and hemolysis, maximally between pH 7 and 8.