van der Goot F G, Ausio J, Wong K R, Pattus F, Buckley J T
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Biol Chem. 1993 Aug 25;268(24):18272-9.
Aerolysin is a channel-forming protein secreted as a protoxin by Aeromonas hydrophila. Analytical centrifugation measurements showed that proaerolysin is a dimer in solution, and this was confirmed by chemical cross-linking with dimethyl suberimidate. Dissociation of proaerolysin with low concentrations of SDS resulted in the loss of tertiary structure, assessed by near ultraviolet circular dichroism. This was accompanied by an increase in the protein's ability to bind the hydrophobic dye 1-anilino-8-naphthalene sulfonate, as well as by increased sensitivity to proteolytic degradation. However, the monomer was not fully unfolded by the detergent, as the tryptophans remained in a hydrophobic environment, and the secondary structure measured by far ultraviolet circular dichroism did not seem to be affected. Aerolysin, the active form of the protein, was also shown to be a dimer, and its stability was found to be no different from the stability of the protoxin dimer. Substituting tryptophan 371 or tryptophan 373 with leucine greatly reduced the stability of dimeric proaerolysin. These substitutions are known to increase the protein's ability to oligomerize, supporting the conclusion that dimer dissociation is necessary for oligomerization to occur.
气单胞菌溶素是嗜水气单胞菌分泌的一种作为原毒素的成孔蛋白。分析超速离心测量表明，原气单胞菌溶素在溶液中是二聚体，用亚胺基二甲酯进行化学交联证实了这一点。用低浓度十二烷基硫酸钠（SDS）解离原气单胞菌溶素会导致三级结构丧失，通过近紫外圆二色性进行评估。这伴随着蛋白质结合疏水染料1-苯胺基-8-萘磺酸盐能力的增加，以及对蛋白水解降解敏感性的增加。然而，去污剂并未使单体完全展开，因为色氨酸仍处于疏水环境中，远紫外圆二色性测量的二级结构似乎未受影响。蛋白质的活性形式气单胞菌溶素也显示为二聚体，并且发现其稳定性与原毒素二聚体的稳定性没有差异。用亮氨酸取代色氨酸371或色氨酸373大大降低了二聚体原气单胞菌溶素的稳定性。已知这些取代会增加蛋白质的寡聚化能力，支持二聚体解离是寡聚化发生所必需的这一结论。
