FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids
FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids
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DOI：
10.1038/nmeth945
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发表时间：
2006-11-01
期刊：
影响因子：
48
通讯作者：
Hohsaka, Takahiro
中科院分区：
文献类型：
--
作者：
Kajihara, Daisuke;Abe, Ryoji;Hohsaka, Takahiro
我们设计并合成了新的、荧光的、非天然的氨基酸，其荧光波长超过500 nm，并被大肠杆菌无细胞翻译系统所接受。我们合成了对氨基苯基丙氨酸衍生物，在对氨基上与BODIPY荧光基团连接，并在无细胞翻译系统中使用四碱基密码子CGGG将其引入链霉亲和素中。实际上，BODIPYFL、BODIPY558和BODIPY576的掺入效率都足够高。接下来，我们将bodipyfl -氨基苯基丙氨酸和bodipy558 -氨基苯基丙氨酸作为两个四碱基密码子的荧光共振能量转移（FRET）的供体和受体对结合到钙调素的不同位置。荧光光谱和偏振测量显示，双标记的钙调素在N端含有BODIPY558，在Gly40、Phe99和Leu112位置含有BODIPYFL，当钙调素结合肽结合时，FRET发生了实质性的变化。这些结果证明了FRET基于位置特异性双结合荧光氨基酸分析蛋白质构象变化的有用性。
We designed and synthesized new, fluorescent, non-natural amino acids that emit fluorescence of wavelengths longer than 500 nm and are accepted by an Escherichia coli cell-free translation system. We synthesized p-aminophenylalanine derivatives linked with BODIPY fluorophores at the p-amino group and introduced them into streptavidin using the four-base codon CGGG in a cell-free translation system. Practically, the incorporation efficiency was high enough for BODIPYFL, BODIPY558 and BODIPY576. Next, we incorporated BODIPYFL-aminophenylalanine and BODIPY558-aminophenylalanine into different positions of calmodulin as a donor and acceptor pair for fluorescence resonance energy transfer (FRET) using two four-base codons. Fluorescence spectra and polarization measurements revealed that substantial FRET changes upon the binding of calmodulin-binding peptide occurred for the double-labeled calmodulins containing BODIPY558 at the N terminus and BODIPYFL at the Gly40, Phe99 and Leu112 positions. These results demonstrate the usefulness of FRET based on the position-specific double incorporation of fluorescent amino acids for analyzing conformational changes of proteins.