Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein.
Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein.
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DOI：
10.1021/bi301105u
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发表时间：
2013-01-29
期刊：
影响因子：
2.9
通讯作者：
Loh SN
中科院分区：
文献类型：
--
作者：
Ha JH;Shinsky SA;Loh SN
交替框架折叠（AFF）是一种蛋白质工程方法，其目的是将普通的结合蛋白转化为分子开关。AFF修饰需要复制蛋白质的氨基或羧基末端片段，并将其附加到分子的另一端。这种复制允许蛋白质以配体依赖的方式在两个相互排斥的天然折叠之间相互转换：野生型结构和环状排列形式。可以通过在对工程化构象变化敏感的位点处放置外来荧光团来检测折叠位移。在这里，我们应用AFF机制创建几个核糖传感蛋白来自腾冲嗜热厌氧杆菌核糖结合蛋白。我们的目的是系统地探索AFF设计的参数。这些考虑因素包括圆形排列的位点、重复片段的长度和位置、开关机制的热力学和动力学优化以及外来荧光团的位置。这里创建的四个AFF变体中的三个经历了预期的构象转变，并表现出核糖依赖性荧光变化。第四种结构在添加核糖后不能转换折叠，可能是因为环状排列形式的折叠比非排列形式的折叠慢得多。这种差异显然引入了一个动力学障碍，分区的重折叠分子的非置换结构。这项研究的结果作为一个指导方针，适用于AFF修改其他蛋白质的生物医学，诊断和工业利益。
Alternate frame folding (AFF) is a protein engineering methodology the purpose of which is to convert an ordinary binding protein into a molecular switch. The AFF modification entails duplicating an amino- or carboxy-terminal segment of the protein and appending it to the opposite end of the molecule. This duplication allows the protein to interconvert, in a ligand-dependent fashion, between two mutually exclusive native folds: the wild-type structure and a circularly permuted form. The fold shift can be detected by placement of extrinsic fluorophores at sites sensitive to the engineered conformational change. Here, we apply the AFF mechanism to create several ribose-sensing proteins derived from Thermoanaerobacter tengcongensis ribose binding protein. Our purpose is to systematically explore the parameters of the AFF design. These considerations include the site of circular permutation, the length and location of the duplicated segment, thermodynamic and kinetic optimization of the switching mechanism, and placement of extrinsic fluorophores. Three of the four AFF variants created here undergo the expected conformational shift and exhibit a ribose-dependent fluorescence change. The fourth construct fails to switch folds upon addition of ribose, likely because the circularly permuted form folds much more slowly than the nonpermuted form. This disparity apparently introduces a kinetic barrier that partitions the refolding molecules to the nonpermuted structure. The results of this study serve as a guideline for applying the AFF modification to other proteins of biomedical, diagnostic, and industrial interest.
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影响因子：
5.6
作者：
Alicea I;Marvin JS;Miklos AE;Ellington AD;Looger LL;Schreiter ER
通讯作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.142089199
发表时间：
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影响因子：
11.1
作者：
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通讯作者：
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DOI：
10.1073/pnas.0907668107
发表时间：
2010-02-16
影响因子：
11.1
作者：
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通讯作者：
Loh, Stewart N.