An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway.
An engineered leucine zipper a position mutant with an unusual three-state unfolding pathway.
复制标题
一种工程亮氨酸拉链的位置突变体，具有不寻常的三态展开途径。
DOI：
10.1110/ps.30901
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Hu,JC
中科院分区：
文献类型：
--
作者：
Zhu,H;Celinski,SA;Scholtz,JM;Hu,JC
亮氨酸拉链是一种二聚体卷曲螺旋结构基序，由四到六个七肽重复组成，指定为 (abcdefg)n。在GCN4亮氨酸拉链中，第三个七联体中的位置16被Asn残基占据，而其他位置是Val残基。最近，我们构建了 GCN4 亮氨酸拉链的变体，其中 aposition Val 残基被 Ile 取代。野生型 GCN4 亮氨酸拉链和 Val 至 Ile 变体的折叠和展开都遵循简单的双态机制。在这项研究中，GCN4亮氨酸拉链的另一个变体是通过将单个Asn残基从位置16移动到位置9来构建的。这种转换导致GCN4亮氨酸拉链的热解折叠变成三态。该变体的解折叠途径通过热变性、有限蛋白酶 K 消化和沉降平衡分析来确定。我们的数据与模型一致，其中变体首先从其 N 末端展开，并将寡聚特异性从天然二聚体改变为包含二聚体和三聚体混合物的部分展开的中间体，然后完全展开为非结构化单体。
The leucine zipper is a dimeric coiled‐coil structural motif consisting of four to six heptad repeats, designated (abcdefg)n. In the GCN4 leucine zipper,aposition 16 in the third heptad is occupied by an Asn residue whereas the otherapositions are Val residues. Recently, we have constructed variants of the GCN4 leucine zipper in which theaposition Val residues were replaced by Ile. The folding and unfolding of the wild‐type GCN4 leucine zipper and the Val to Ile variant both adhere to a simple two‐state mechanism. In this study, another variant of the GCN4 leucine zipper was constructed by moving the single Asn residue fromaposition 16 toaposition 9. This switch causes the thermal unfolding of the GCN4 leucine zipper to become three state. The unfolding pathway of this variant was determined by thermal denaturation, limited proteinase K digestion, and sedimentation equilibrium analysis. Our data are consistent with a model in which the variant first unfolds from its N terminus and changes the oligomerization specificity from a native dimer to a partially unfolded intermediate containing a mixture of dimers and trimers and then completely unfolds to unstructured monomers.