Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay |
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Authors: | Eun-Ju Jeong Kyoungsook Park Hyou-Arm Joung Chang-Soo Lee Dai-Wu Seol Bong Hyun Chung Moonil Kim |
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Institution: | (1) BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon, 305-600, Republic of Korea;(2) Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA |
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Abstract: | Conformational changes in hexokinase are induced by its binding to glucose, thus providing an excellent example of an ‘induced
fit’ model. To observe glucose-induced fluorescence restoration in hexokinase II using split-enhanced, green fluorescent protein
(EGFP) in a process involving the reconstitution of split EGFP, E. coli cells expressing the chimeric NEGFP:HXK:CEGFP recombinant protein were treated with glucose and visualized via fluorescence
read-outs. The reconstituted EGFP generated a strong fluorescence upon glucose stimulation of the bacteria. Moreover, the
fluorescence intensity became stronger with increasing glucose up to 10 mM, with a maximum being observed after 60 min in
a time- and concentration-dependent manner. Conformational changes associated with glucose-induced fit in human hexokinase
II can thus be monitored successfully in vivo via fluorescence reconstitution assays, coupled with a quick and easy fluorescent
read-out protocol. |
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Keywords: | Conformational change Fluorescence complementation assay Glucose-induced fit Hexokinase II Split EGFP |
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