Ritco-Vonsovici M, Minard P, Desmadril M, Yon J M
Laboratoire d'enzymologie physicochimique et moléculaire, Centre National de la Recherche Scientifique, Université de Paris-Sud, Orsay, France.
Biochemistry. 1995 Dec 26;34(51):16543-51. doi: 10.1021/bi00051a002.
The role of domains in protein folding has been widely studied and discussed. Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme [Minard, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Protein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams, R. J. P. (1989) Eur. J. Biochem. 184, 617-625; Missiakas, D., Betton, J. M., Minard, P., & Yon, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gene were constructed. The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively. These two proteins were overexpressed and purified. They exhibit 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules. The unfolding studies reveal a large decrease in stability. From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure.
结构域在蛋白质折叠过程中的作用已得到广泛研究和讨论。然而，目前尚不清楚蛋白质中结构域的连续性是否是决定折叠途径的必要条件。先前的研究表明，双结构域单体酶酵母磷酸甘油酸激酶（yPGK）的分离结构域能够独立折叠成准天然结构，但它们既不会重新结合也不会产生有功能的酶[米纳德，P.，霍尔，L.，贝顿，J.M.，米西亚卡斯，D.，&扬，J.M.（1989年）《蛋白质工程》3，55 - 60；费尔布拉泽，W.J.，鲍文，D.，霍尔，L.，威廉姆斯，R.J.P.（1989年）《欧洲生物化学杂志》184，617 - 625；米西亚卡斯，D.，贝顿，J.M.，米纳德，P.，&扬，J.M.（1990年）《生物化学》29，8683 - 8689]。在本研究中，构建了yPGK基因的两个环形排列变体。天然相邻的链端被直接连接，新的末端分别在N结构域（第71和72位残基处）或C结构域（第291和292位残基处）内产生。这两种蛋白质被过量表达并纯化。它们分别表现出野生型酶活性的14%和23%。这两个突变体以紧密构象折叠，二级和三级结构略有变化，这可能与分子的异质性群体的存在有关。去折叠研究表明稳定性大幅下降。从目前的数据来看，虽然环形排列在蛋白质的结构和稳定性上引起了一些扰动，但结构域的连续性并非蛋白质达到类似天然且有功能结构所必需的。
