Yokota A, Izutani K, Takai M, Kubo Y, Noda Y, Koumoto Y, Tachibana H, Segawa S
Department of Physics, School of Science, Kwansei Gakuin University, Nishinomiya, 662-8501, Japan.
J Mol Biol. 2000 Feb 4;295(5):1275-88. doi: 10.1006/jmbi.1999.3442.
The effects of lacking a specific disulfide bridge on the transition state in folding were examined in order to explore the folding-unfolding mechanism of lysozyme. Four species of three-disulfide variant of hen lysozyme (3SS-lysozyme) were prepared by replacing two Cys residues with Ala or Ser: C6S/C127A, C30A/C115A, C64A/C80A and C76A/C94A. The recombinant hen lysozyme was studied as the standard reference containing four authentic disulfide bridges and the extra N-terminal Met: the recombinant hen lysozyme containing the extra N-terminal. Folding rates were measured by monitoring the change in fluorescence intensity associated with tri-N-acetyl-d-glucosamine binding to the active site of refolded lysozyme. It was confirmed that the folding rate of the recombinant hen lysozyme containing the extra N-terminal was the same as that of wild-type lysozyme, and that the folding rate was little affected by the presence of tri-N-acetyl-d-glucosamine (triNAG). The folding rate of C64A/C80A was found to be the fastest and almost the same as that of the recombinant hen lysozyme containing the extra N-terminal, and that of C30A/C115A the second, and that of C6S/C127A the third. The folding rate of C76A/C94A was particularly slow. On the other hand, the unfolding rates which were measured in the presence of triNAG showed the dependence on the concentration of triNAG. The intrinsic unfolding rate in the absence of triNAG was determined by extrapolation. Also in the unfolding rate, C76A/C94A was markedly slower than the others. It was found from the analysis of binding constants of triNAG to C64A/C80A during the unfolding process that the active site of C64A/C80A partly unfolds already prior to the unfolding transition. On the basis of these kinetic data, we suggest that C64A/C80A folding transition can occur with leaving the loop region around SS3 (C64-C80) flexible, while cross-linking by SS4 (C76-C94) is important for the promotion of folding, because it is an indispensable constraint on the way towards the folding transition state.
为了探究溶菌酶的折叠-去折叠机制，研究了缺少特定二硫键对折叠过渡态的影响。通过将两个半胱氨酸残基替换为丙氨酸或丝氨酸，制备了四种三硫键变体的母鸡溶菌酶（3SS-溶菌酶）：C6S/C127A、C30A/C115A、C64A/C80A和C76A/C94A。以含有四个天然二硫键和额外N端甲硫氨酸的重组母鸡溶菌酶作为标准参考进行研究：含有额外N端的重组母鸡溶菌酶。通过监测与三-N-乙酰-d-葡萄糖胺结合到复性溶菌酶活性位点相关的荧光强度变化来测量折叠速率。证实含有额外N端的重组母鸡溶菌酶的折叠速率与野生型溶菌酶相同，并且折叠速率几乎不受三-N-乙酰-d-葡萄糖胺（triNAG）存在的影响。发现C64A/C80A的折叠速率最快，几乎与含有额外N端的重组母鸡溶菌酶相同，C30A/C115A的折叠速率次之，C6S/C127A的折叠速率排第三。C76A/C94A的折叠速率特别慢。另一方面，在triNAG存在下测量的去折叠速率显示出对triNAG浓度的依赖性。通过外推确定了不存在triNAG时的固有去折叠速率。在去折叠速率方面，C76A/C94A也明显比其他变体慢。通过分析去折叠过程中triNAG与C64A/C80A的结合常数发现，C64A/C80A的活性位点在去折叠转变之前已经部分展开。基于这些动力学数据，我们认为C64A/C80A的折叠转变可以在使SS3（C64-C80）周围的环区域保持柔性的情况下发生，而SS4（C76-C94）的交联对于促进折叠很重要，因为它是通向折叠过渡态过程中不可或缺的限制因素。
