TY - JOUR
T1 - Construction of a 'turn-on' fluorescent probe system for His-tagged proteins
AU - Murata, Atsushi
AU - Arai, Satoshi
AU - Yoon, Su In
AU - Takabayashi, Masao
AU - Ozaki, Miwako
AU - Takeoka, Shinji
N1 - Funding Information:
The authors thank Dr. Shin’ichi Ishiwata, Dr. Madoka Suzuki (Waseda University) and Dr. Lu Yixin (National University of Singapore) for useful discussion. This work was partially supported by Special Coordination Funds for Promoting Science and Technology ‘Development of the advanced techniques for imaging molecular dynamics at various levels of hierarchy’ to S.T., M.O. and S.A., Global COE ‘Practical Chemical Wisdom’ to A.M., and ‘High-Tech Research Center’ project to S.T. from MEXT, Japan.
PY - 2010/12/1
Y1 - 2010/12/1
N2 - Hexahistidine ((His)6) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)6, and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)6. The system is turned off when Dabcyl-(His)6 is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)6-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)6-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His) 6-protein.
AB - Hexahistidine ((His)6) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)6, and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni2+, can bind (His)6. The system is turned off when Dabcyl-(His)6 is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)6-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)6-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His) 6-protein.
KW - Energy transfer
KW - Fluorescent probe
KW - Histidine-tag
KW - Molecular recognition
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U2 - 10.1016/j.bmcl.2010.10.011
DO - 10.1016/j.bmcl.2010.10.011
M3 - Article
C2 - 21035333
AN - SCOPUS:78149286156
VL - 20
SP - 6905
EP - 6908
JO - Bioorganic and Medicinal Chemistry Letters
JF - Bioorganic and Medicinal Chemistry Letters
SN - 0960-894X
IS - 23
ER -