Ha Jeung-Hoi, Loh Stewart N
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY, USA.
Methods Mol Biol. 2017;1596:27-41. doi: 10.1007/978-1-4939-6940-1_2.
Alternate frame folding (AFF) and protein/fragment exchange (FREX) are related technologies for engineering allosteric conformational changes into proteins that have no pre-existing allosteric properties. One of their chief purposes is to turn an ordinary protein into a biomolecular switch capable of transforming an input event into an optical or functional readout. Here, we present a guide for converting an arbitrary binding protein into a fluorescent biosensor with Förster resonance energy transfer output. Because the AFF and FREX mechanisms are founded on general principles of protein structure and stability rather than a property that is idiosyncratic to the target protein, the basic design steps-choice of permutation/cleavage sites, molecular biology, and construct optimization-remain the same for any target protein. We highlight effective strategies as well as common pitfalls based on our experience with multiple AFF and FREX constructs.
交替框架折叠（AFF）和蛋白质/片段交换（FREX）是用于将变构构象变化工程化到原本没有变构特性的蛋白质中的相关技术。它们的主要目的之一是将普通蛋白质转变为能够将输入事件转化为光学或功能读数的生物分子开关。在此，我们提供了一份将任意结合蛋白转化为具有Förster共振能量转移输出的荧光生物传感器的指南。由于AFF和FREX机制基于蛋白质结构和稳定性的一般原则，而非目标蛋白质特有的属性，对于任何目标蛋白质，基本设计步骤——排列/切割位点的选择、分子生物学和构建体优化——都是相同的。基于我们对多个AFF和FREX构建体的经验，我们突出了有效的策略以及常见的陷阱。
