A Ca2+-sensing molecular switch based on alternate frame protein folding.
A Ca2+-sensing molecular switch based on alternate frame protein folding.
复制标题
DOI：
10.1021/cb800177f
复制
发表时间：
2008-11-21
影响因子：
4
通讯作者：
Loh, Stewart N.
中科院分区：
文献类型：
--
作者：
Stratton, Margaret M.;Mitrea, Diana M.;Loh, Stewart N.
现有的制造生物传感器的策略主要依赖于大的构象变化来将结合事件转化为输出信号。然而，大多数分子在底物结合后不会表现出大规模的结构变化。在这里，我们提出了一种通用的方法（交替框架折叠，或AFF）工程变构控制到配体结合蛋白。原则上，AFF可以应用于任何蛋白质以建立结合诱导的构象变化，即使天然分子中不存在构象变化。AFF设计复制了一部分氨基酸序列，创造了一个额外的折叠“框架”。一个框架对应于野生型序列，折叠产生正常结构。在第二帧中折叠产生环状排列的蛋白质。由于这两种天然结构竞争共享序列，它们以互斥的方式折叠。结合能被用来驱动构象从一个折叠到另一个折叠的变化。我们通过将钙结合蛋白D9k转化为感知Ca2+的分子开关来证明这种方法。Ca2+游离和Ca2+结合的钙结合蛋白的结构几乎相同。然而，AFF机制设计了一个强大的构象变化，我们使用两个共价连接的荧光基团检测。生物荧光团也可以用来制造基因编码的传感器。AFF应广泛适用于制造各种小分子的传感器。
Existing strategies for creating biosensors mainly rely on large conformational changes to transduce a binding event to an output signal. Most molecules, however, do not exhibit large-scale structural changes upon substrate binding. Here, we present a general approach (alternate frame folding, or AFF) for engineering allosteric control into ligand binding proteins. AFF can in principle be applied to any protein to establish a binding-induced conformational change, even if none exists in the natural molecule. The AFF design duplicates a portion of the amino acid sequence, creating an additional “frame” of folding. One frame corresponds to the wild-type sequence, and folding produces the normal structure. Folding in the second frame yields a circularly permuted protein. Because the two native structures compete for a shared sequence, they fold in a mutually exclusive fashion. Binding energy is used to drive the conformational change from one fold to the other. We demonstrate the approach by converting the protein calbindin D9k into a molecular switch that senses Ca2+. The structures of Ca2+-free and Ca2+-bound calbindin are nearly identical. Nevertheless, the AFF mechanism engineers a robust conformational change that we detect using two covalently attached fluorescent groups. Biological fluorophores can also be employed to create a genetically encoded sensor. AFF should be broadly applicable to create sensors for a variety of small molecules.
登录
查看更多内容
影响因子：
5.6
作者：
CARLSTROM, G;CHAZIN, WJ
通讯作者：
CHAZIN, WJ
DOI：
10.1002/prot.10080
发表时间：
2002-05-15
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
Julenius, K;Robblee, J;Linse, S
通讯作者：
Linse, S
影响因子：
56.9
作者：
LUGER, K;HOMMEL, U;KIRSCHNER, K
通讯作者：
KIRSCHNER, K
影响因子：
2.9
作者：
EDELHOCH, H
通讯作者：
EDELHOCH, H
影响因子：
5.8
作者：
Paszkiewicz, Konrad H.;Sternberg, Michael J. E.;Lappe, Michael
通讯作者：
Lappe, Michael