Zavialov A, Benndorf R, Ehrnsperger M, Zav'yalov V, Dudich I, Buchner J, Gaestel M
Institute of Immunological Engineering, Moscow Region, Russian Federation.
Int J Biol Macromol. 1998 May-Jun;22(3-4):163-73. doi: 10.1016/s0141-8130(98)00014-2.
The murine small heat shock protein Hsp25 carries a single cysteine residue in position 141 of its amino acid sequence. Interestingly, Hsp25 can exist within the cell as covalently bound dimer which is linked by an intermolecular disulfide bond between two monomers. Oxidative stress caused by treatment of the cells with diamide, arsenite, or hydrogen peroxide leads to an increase in Hsp25-dimerisation which can be blocked by simultaneous treatment with reducing agents. Recombinant Hsp25 was prepared in an oxidized dimeric (oxHsp25) and reduced monomeric (redHsp25) from. The two species were compared with regard to secondary structure, stability, oligomerization properties and their chaperone activity. It is demonstrated by CD measurements in the far UV region that there are no significant differences in the secondary structure and temperature- or pH-stability of oxHsp25 and redHsp25. However, according to CD measurements in the near UV region an increase in the asymmetry of the microenvironment of aromatic residues in oxHsp25 is observed. Furthermore, an increase in stability of the hydrophobic environment of the tryptophan residues mainly located in the N-terminal domain of the protein against urea denaturation is detected in oxHsp25. Both reduced and oxidized Hsp25 from oligomeric complexes of similar size and stability against detergents and both species prevent thermal aggregation of citrate synthase and assist significantly in oxaloacetic acid-induced refolding of the enzyme. Hence, the overall secondary structure, the degree of oligomerization and the chaperone activity of Hsp25 seem independent of the formation of the intermolecular disulfide bond and only the stability of the hydrophobic N-terminal part of the molecule is influenced by formation of this bound. The obtained data do not exclude the possible involvement of dimerization of this protein in other cellular functions, e.g. in intracellular sulfhydryl-buffering or in the protection of actin filaments from fragmentation upon oxidative stress.
小鼠小分子热休克蛋白Hsp25在其氨基酸序列的第141位带有一个半胱氨酸残基。有趣的是，Hsp25在细胞内可以以共价结合的二聚体形式存在，该二聚体通过两个单体之间的分子间二硫键相连。用二酰胺、亚砷酸盐或过氧化氢处理细胞所引起的氧化应激会导致Hsp25二聚化增加，而同时用还原剂处理则可阻断这种增加。重组Hsp25以氧化二聚体（oxHsp25）和还原单体（redHsp25）形式制备。比较了这两种形式在二级结构、稳定性、寡聚化特性及其伴侣活性方面的差异。远紫外区域的圆二色性（CD）测量表明，oxHsp25和redHsp25在二级结构以及温度或pH稳定性方面没有显著差异。然而，根据近紫外区域的CD测量，观察到oxHsp25中芳香族残基微环境的不对称性增加。此外，在oxHsp25中检测到主要位于蛋白质N端结构域的色氨酸残基的疏水环境对尿素变性的稳定性增加。还原型和氧化型Hsp25形成大小和稳定性相似的寡聚复合物，且两种形式都能防止柠檬酸合酶的热聚集，并显著协助草酰乙酸诱导的该酶重折叠。因此，Hsp2�的整体二级结构、寡聚化程度和伴侣活性似乎与分子间二硫键的形成无关，只有分子疏水N端部分的稳定性受该键形成的影响。所获得的数据并不排除该蛋白二聚化可能参与其他细胞功能，例如细胞内巯基缓冲或在氧化应激时保护肌动蛋白丝不发生断裂。
