Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
Bioconjug Chem. 2009 Dec;20(12):2324-30. doi: 10.1021/bc900330w.
This study demonstrates a unique, nontranscriptional assay system based on molecular tension of a luciferase artificially appended by protein-protein binding. We hypothesized that an artificially appended molecular tension to a full-length luciferase may diversify the enzymatic activity through a modification of the active site. For the basic probe design, a full-length luciferase was sandwiched between two component proteins of interest. The length of flexible linkers between the components was minimized to exert an efficient molecular tension to the sandwiched luciferase. When N- and C-terminal ends of Renilla luciferase 8 were flanked by the ligand-binding domain of human estrogen receptor alpha (ER LBD) and SH2 domain of Src, named ERS, this simple probe was surprisingly sensitive to estrogens. The luminescence spectra by ERS were largely enhanced by an addition of 4-hydroxytamoxifen (OHT), 17beta-estradiol, and genistein. The detection limit of ERS reached 1 nM OHT. Quantum yield (QY) and Michaelis-Menten constant of ERS were found to be 6.3% and 94.3 muM, respectively. The enzymatic activities of ERS are also governed by different types of coelenterazine (CTZ) variants. The two hydroxy groups in CTZ are critical for the enzymatic activities of ERS. This study is the first example that an artificially appended molecular tension to a full-length luciferase can be taken as an optical signature upon molecular imaging. This study also provides new insight into the construction of a new lineage of bioluminescent probes for estimating protein-protein interactions.
本研究展示了一种独特的、基于荧光素酶分子张力的非转录测定系统，该荧光素酶通过蛋白-蛋白结合人工附加。我们假设，通过对活性位点的修饰，人工附加于全长荧光素酶的分子张力可能会使酶活性多样化。对于基本探针设计，全长荧光素酶被夹在两个感兴趣的成分蛋白之间。组件之间的柔性接头长度被最小化，以对夹心荧光素酶施加有效的分子张力。当海肾荧光素酶 8 的 N 和 C 末端被人雌激素受体α（ER LBD）的配体结合域和Src 的 SH2 结构域侧翼时，命名为 ERS，这个简单的探针对雌激素出乎意料地敏感。ERS 的发光光谱通过添加 4-羟基他莫昔芬（OHT）、17β-雌二醇和染料木黄酮大大增强。ERS 的检测限达到 1 nM OHT。ERS 的量子产率（QY）和米氏常数分别为 6.3%和 94.3 μM。ERS 的酶活性也受不同类型的海肾发光蛋白（CTZ）变体的控制。CTZ 中的两个羟基对于 ERS 的酶活性至关重要。本研究首次证明，人工附加于全长荧光素酶的分子张力可作为分子成像的光学特征。本研究还为构建新的生物发光探针以估计蛋白质-蛋白质相互作用提供了新的见解。
