Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118
Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118
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DOI：
10.1093/oxfordjournals.jbchem.a022747
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发表时间：
2000-08-01
影响因子：
2.7
通讯作者：
Yamada, H
中科院分区：
文献类型：
--
作者：
Futami, J;Tada, H;Yamada, H
为了通过引入分子内交联剂来稳定人RNase1，设计了一种突变蛋白(4-118CL RNase1)，其中Arg4和Val118被半胱氨酸残基取代，并以二硫键连接在一起。在氧化还原条件下复性的4-118CL RNase1是一种没有游离SH基团的单体，保持了野生型重组RNase1 11%的活性，表明突变的RNase1正确折叠，并在Cys4和Cys118之间形成了一个额外的二硫键。这一数值低于从理论上计算的5.4千卡/摩尔，这是由于引入了交联键而导致的未折叠状态的链熵降低。这些结果表明，目前的交联剂也使RNase1的折叠状态不稳定3.4kcal/mol。随着热力学稳定性的提高，这种交联剂的引入也显著提高了核糖核酸酶1对胰酶消化的稳定性。稳定的人类核糖核酸酶很可能在临床上是有利的，因为基于核糖核酸酶的免疫毒素在内吞作用后对蛋白质的降解应该有很长的半衰期。
In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118, From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.