An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.
An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.
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具有天然样结构的酵母磷酸甘油酸激酶的工程氨基末端结构域。
DOI：
10.1002/pro.5560060415
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Mas,MT
中科院分区：
文献类型：
--
作者：
Sherman,MA;Chen,Y;Mas,MT
先前的研究表明，酵母磷酸甘油酸激酶的羧基末端肽（残基401-415）和结构域间螺旋（残基185-199）在氨基末端结构域（残基1-184）的折叠和稳定性中起作用。在缺失突变体中，羧基末端肽通过柔性肽连接物连接到氨基末端结构域（残基1-184）和结构域间螺旋（残基185-199），从而完全消除了羧基末端结构域。CD、荧光、凝胶过滤和核磁共振实验表明，与之前描述的不同，这种分离的N -结构域是可溶的、单体的、折叠紧凑的、结构类似天然的，并且能够以饱和的方式以高亲和力结合底物3 -磷酸甘油酸。胍诱导的展开转变的中点与天然两结构域蛋白的中点相同（Cm ~ 0.8 M）。与胍诱导的展开相关的自由能变化是天然酶的三分之一，这与先前评估N -结构域的内在稳定性和结构域相互作用对PGK稳定性的贡献的研究一致。这些观察结果表明，C末端肽和结构域间螺旋足以在缺乏C结构域的情况下维持N结构域的天然折叠。
Previous studies have suggested that the carboxy‐terminal peptide (residues 401–415) and interdomain helix (residues 185–199) of yeast phosphoglycerate kinase, a two‐domain enzyme, play a role in the folding and stability of the amino‐terminal domain (residues 1–184). A deletion mutant has been created in which the carboxy‐terminal peptide is attached to the amino‐terminal domain (residues 1–184) plus interdomain helix (residues 185–199) through a flexible peptide linker, thus eliminating the carboxy‐terminal domain entirely. CD, fluorescence, gel filtration, and NMR experiments indicated that, unlike versions described previously, this isolated N‐domain is soluble, monomeric, compactly folded, native‐like in structure, and capable of binding the substrate 3‐phosphoglycerate with high affinity in a saturable manner. The midpoint of the guanidine‐induced unfolding transition was the same as that of the native two‐domain protein (Cm∼ 0.8 M). The free energy change associated with guanidine‐induced unfolding was one‐third that of the native enzyme, in agreement with previous studies that evaluated the intrinsic stability of the N‐domain and the contribution of domain‐domain interactions to the stability of PGK. These observations suggest that the C‐terminal peptide and interdomain helix are sufficient for maintaining a native‐like fold of the N‐domain in the absence of the C‐domain.