Kim Sung Bae, Nishihara Ryo, Citterio Daniel, Suzuki Koji
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST) , 16-1 Onogawa, Tsukuba 305-8569, Japan.
Department of Applied Chemistry, Faculty of Science and Technology, Keio University , 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan.
Bioconjug Chem. 2016 Feb 17;27(2):354-62. doi: 10.1021/acs.bioconjchem.5b00421. Epub 2015 Sep 30.
Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.
蛋白质-蛋白质相互作用（PPI）的光学成像有助于全面阐明细胞内分子事件。我们展示了一种光学方法，用于可视化哺乳动物细胞中任何PPI触发的分子张力。制作了23种候选设计，其中全长人工荧光素酶（ALuc）夹在两种感兴趣的模型蛋白之间，例如FKBP和FRB。其中一种设计在响应不同浓度的雷帕霉素时极大地增强了生物发光。通过阴性对照证实，升高的生物发光仅由分子张力引起。对探针设计进行了进一步修改以去除ALuc的C末端，发现这提高了信噪比，命名为“组合探针”。通过详细的底物选择性、活细胞的生物发光成像和不同的PPI模型阐明了其效用。本研究扩展了荧光素酶作为分析活体中分子动力学和细胞信号传导工具的能力。
