Robinson A S, King J
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Nat Struct Biol. 1997 Jun;4(6):450-5. doi: 10.1038/nsb0697-450.
The trimeric parallel beta-coil P22 tailspike contains eight cysteines per chain, but lacks disulphide bonds in the native state, in both the crystalline and solution forms. However, cysteines in a folding intermediate are reactive with thiol blocking reagents, which prevent further productive folding both in vivo and in vitro. The in vivo refolding yield was independent of the availability of metal ions, but was sensitive to redox potential. Isolation by nondenaturing gel electrophoresis of the protrimer intermediate, a trimeric folding intermediate that precedes the fully folded trimer in the in vivo and in vitro pathways, revealed the presence of interchain disulphide bonds. Incubation of the isolated protrimer with reducing agents generated the native trimer. The formation of beta-sheets with interdigitated strands from different subunits in the native trimer may require the transient disulphide bonds for proper alignment. To our knowledge this is the first report of a disulphide bond present in a folding intermediate of a non-disulphide bonded protein.
三聚体平行β-螺旋P22尾刺蛋白每条链含有8个半胱氨酸，但在天然状态下，无论是晶体形式还是溶液形式，均不存在二硫键。然而，折叠中间体中的半胱氨酸可与硫醇封闭试剂发生反应，这在体内和体外均会阻止进一步的有效折叠。体内重折叠产率与金属离子的可用性无关，但对氧化还原电位敏感。通过非变性凝胶电泳分离前三聚体中间体（一种在体内和体外途径中先于完全折叠的三聚体的三聚体折叠中间体），发现存在链间二硫键。将分离的前三聚体与还原剂一起孵育可生成天然三聚体。天然三聚体中来自不同亚基的具有相互交错链的β-折叠片层的形成可能需要瞬时二硫键来进行正确排列。据我们所知，这是关于非二硫键结合蛋白折叠中间体中存在二硫键的首次报道。
