Variants of ribonuclease inhibitor that resist oxidation.
Variants of ribonuclease inhibitor that resist oxidation.
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抗氧化的核糖核酸酶抑制剂的变体。
DOI：
10.1110/ps.8.2.430
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Raines,RT
中科院分区：
文献类型：
--
作者：
Kim,BM;Schultz,LW;Raines,RT
人核糖核酸酶抑制因子（Human ribonuclease inhibitor，hRI）是一种保护细胞免受胰腺型核糖核酸酶侵袭的胞浆蛋白。hRI具有32个半胱氨酸残基。这些半胱氨酸残基氧化形成二硫键是一个快速的协同过程，使hRI失活。天然hRI中最近端的半胱氨酸残基是序列上相邻的两对：Cys 94和Cys 95，以及Cys 328和Cys 329。由这种相邻的半胱氨酸残基形成的胱氨酸可能在其八元环内含有干扰性顺式肽键，这将破坏hRI的结构并可能促进进一步氧化。我们发现，用丙氨酸残基取代Cys 328和Cys 329对hRI对牛胰腺核糖核酸酶A（RNase A）的亲和力几乎没有影响，但其抗氧化性增加了10至15倍。对于单一变体C328 A hRI和C329 A hRI观察到类似的效果，表明抗氧化性源于不能形成Cys 328-Cys 329二硫键。用丙氨酸残基取代Cys 94和Cys 95在较小程度上增加了抗氧化性，并降低了hRI对RNA酶A的亲和力。C328 A、C329 A和C328 A/C329 A变体可能比野生型hRI在体外和体内抑制胰腺型核糖核酸酶方面更有用。我们的结论是，取代相邻的半胱氨酸残基可以赋予蛋白质的抗氧化性。
Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328–Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.