Peptide rescue of an N‐terminal truncation of the stoffel fragment of Taq DNA polymerase
Peptide rescue of an N‐terminal truncation of the stoffel fragment of Taq DNA polymerase
复制标题
Taq DNA 聚合酶 stoffel 片段 N 端截短的肽拯救
作者：
I. Vainshtein;B. Malcolm;A. Atrazhev;J. Elliott;S. Eom;D. Wishart
来自水生栖热菌的DNA聚合酶（Taq聚合酶）的前289个氨基酸的缺失去除了5′至3′外切核酸酶结构域以产生热稳定的斯托费尔聚合酶片段（Lawyer等人，1989年）。斯托费尔片段的初步N末端截短研究表明，去除额外的12个氨基酸（StofΔ12突变体）对活性或稳定性没有显著影响，但蛋白质的进一步截短（StofΔ47，其中47个氨基酸被删除）导致活性和热稳定性的显著损失。一种33个氨基酸的合成肽，基于这个关键区域（即，残基303-335（包括端点），当加回截短的StofΔ47蛋白时，能够恢复85%的StofΔ12活性，并将最适温度恢复到StofΔ12和斯托费尔蛋白的最适温度。Taq聚合酶的晶体结构的检查（Kim等人，1995）显示酶的残基302-336形成与蛋白质的其余部分相互作用的三链β折叠结构。33个氨基酸肽的CD分析表明，游离肽在溶液中也采用了有序结构，β折叠含量超过50%。这些数据表明，这种33个氨基酸的肽构成了稳定的β折叠结构，能够以类似于充分记录的核糖核酸酶S蛋白通过15个残基的α螺旋S肽的互补的方式拯救截短的聚合酶。
Deletion of the first 289 amino acids of the DNA polymerase from Thermus aquaticus (Taq polymerase) removes the 5′ to 3′ exonuclease domain to yield the thermostable Stoffel polymerase fragment (Lawyer et al., 1989). Preliminary N‐terminal truncation studies of the Stoffel fragment suggested that removal of an additional 12 amino acids (the StofΔ12 mutant) had no significant effect on activity or stability, but that the further truncation of the protein (the StofΔ47, in which 47 amino acids were deleted), resulted in a significant loss of both activity and thermostability. A 33‐amino acid synthetic peptide, based on this critical region (i.e., residues 303–335 inclusive), was able to restore 85% of the StofΔ12 activity when added back to the truncated StofΔ47 protein as well as return the temperature optimum to that of the StofΔ12 and Stoffel proteins. Examination of the crystal structure of Taq polymerase (Kim et al., 1995) shows that residues 302–336 of the enzyme form a three‐stranded β‐sheet structure that interacts with the remainder of the protein. CD analysis of the 33‐amino acid peptide indicates that the free peptide also adopts an ordered structure in solution with more than 50% β‐sheet content. These data suggest that this 33‐amino acid peptide constitutes a stable β‐sheet structure capable of rescuing the truncated polymerase in a fashion analogous to the well‐documented complementation of Ribonuclease S protein by the 15‐residue, α‐helical, S peptide.
影响因子：
56.9
作者：
SCHAFMEISTER, CE;MIERCKE, LJW;STROUD, RM
通讯作者：
STROUD, RM
影响因子：
2.9
作者：
ENGELKE, DR;KRIKOS, A;GINSBURG, D
通讯作者：
GINSBURG, D