title: Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay.
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.
authors.affiliation: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon, 305-600, Republic of Korea.
authors.forename: Eun-Ju
authors.initials: EJ
authors.lastname: Jeong
authors.forename: Kyoungsook
authors.initials: K
authors.lastname: Park
authors.forename: Hyou-Arm
authors.initials: HA
authors.lastname: Joung
authors.forename: Chang-Soo
authors.initials: CS
authors.lastname: Lee
authors.forename: Dai-Wu
authors.initials: DW
authors.lastname: Seol
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Chung
authors.forename: Moonil
authors.initials: M
authors.lastname: Kim
mesh_terms: D001681:Biological Assay / Q000379:methods*; D005453:Fluorescence; D005947:Glucose / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006593:Hexokinase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011487:Protein Conformation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006593:Hexokinase; D005947:Glucose