Stratton Margaret M, Mitrea Diana M, Loh Stewart N
Department of Biochemistry & Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, New York 13210, USA.
ACS Chem Biol. 2008 Nov 21;3(11):723-32. doi: 10.1021/cb800177f.
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 D(9k) 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.
现有的生物传感器构建策略主要依赖于大规模的构象变化，以将结合事件转化为输出信号。然而，大多数分子在与底物结合时并不会表现出大规模的结构变化。在此，我们提出了一种通用方法（交替框架折叠，即AFF），用于在配体结合蛋白中引入变构控制。原则上，AFF可应用于任何蛋白质，以建立结合诱导的构象变化，即使天然分子中不存在这种变化。AFF设计复制了一部分氨基酸序列，创建了一个额外的折叠“框架”。一个框架对应野生型序列，折叠产生正常结构。第二个框架中的折叠产生一个环形排列的蛋白质。由于这两种天然结构争夺共享序列，它们以互斥的方式折叠。结合能用于驱动从一种折叠到另一种折叠的构象变化。我们通过将钙结合蛋白D（9k）转化为一种感知Ca2+的分子开关来证明该方法。无Ca2+和结合Ca2+的钙结合蛋白的结构几乎相同。然而，AFF机制设计了一种强大的构象变化，我们使用两个共价连接的荧光基团来检测这种变化。生物荧光团也可用于创建基因编码传感器。AFF应该广泛适用于创建针对各种小分子的传感器。
