Engineering an artificial zymogen by alternate frame protein folding
Engineering an artificial zymogen by alternate frame protein folding
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DOI：
10.1073/pnas.0907668107
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发表时间：
2010-02-16
影响因子：
11.1
通讯作者：
Loh, Stewart N.
中科院分区：
文献类型：
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作者：
Mitrea, Diana M.;Parsons, Lee S.;Loh, Stewart N.
交替框架折叠（AFF）是一种新的机制，通过它可以将变构引入蛋白质中，而以前可能不存在。我们采用这种技术将细胞毒性核糖核酸酶芽孢杆菌RNA酶转化为人工酶原，该酶原由HIV-1蛋白酶激活。AFF修饰需要多肽链的部分复制和复制片段之一中的关键催化残基的突变。所得分子可以在两个“框架”中的一个中折叠以产生野生型结构或环状排列形式，其中N-和C-末端的位置与表面环交换。它不能同时具有两种结构，因为每种结构都会竞争共享的氨基酸序列。一个HIV-1蛋白酶识别序列被插入到非排列框架的一个表面环中，切割诱导从非排列折叠到排列折叠的转变。使用AFF机制，相对于野生型芽孢杆菌RNA酶，我们能够在酶原中抑制k（cat）/K-M 250倍。HIV-1蛋白酶切割随后使k（cat）/K-M增加130倍。AFF是重要的，因为它是通用的，原则上可以用于控制许多酶的活性，包括那些功能不受任何现有机制调节的酶。
Alternate frame folding (AFF) is a novel mechanism by which allostery can be introduced into a protein where none may have existed previously. We employ this technology to convert the cytotoxic ribonuclease barnase into an artificial zymogen that is activated by HIV-1 protease. The AFF modification entails partial duplication of the polypeptide chain and mutation of a key catalytic residue in one of the duplicated segments. The resulting molecule can fold in one of two "frames" to yield the wild-type structure or a circularly permuted form in which the positions of the N- and C-termini are exchanged with a surface loop. It cannot take on both structures simultaneously because each competes for a shared amino acid sequence. An HIV-1 protease recognition sequence is inserted into one of the surface loops in the nonpermuted frame, and cleavage induces a shift from the nonpermuted fold to the permuted fold. Using the AFF mechanism, we were able to suppress k(cat)/K-M by 250-fold in the proenzyme relative to wild-type barnase. HIV-1 protease cleavage subsequently increases k(cat)/K-M by 130-fold. AFF is significant because it is general and can in principle be used to control activity of many enzymes, including those whose functions are not regulated by any existing mechanism.