Molecular tension-indexed bioluminescent probe for determining protein-protein interactions.
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.
Kim
Sung Bae
SB
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
Sato
Moritoshi
M
Tao
Hiroaki
H
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D047628:Estrogen Receptor alpha / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000378:metabolism*; D049408:Luminescent Agents / Q000032:analysis* / Q000737:chemistry; D008163:Luminescent Measurements; D015394:Molecular Structure; D011485:Protein Binding; D013500:Surface Tension; D018909:src Homology Domains
D047628:Estrogen Receptor alpha; D008024:Ligands; D049408:Luminescent Agents; D049410:Luciferases, Renilla