Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay.
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.
Jeong
Eun-Ju
EJ
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon, 305-600, Republic of Korea.
Park
Kyoungsook
K
Joung
Hyou-Arm
HA
Lee
Chang-Soo
CS
Seol
Dai-Wu
DW
Chung
Bong Hyun
BH
Kim
Moonil
M
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
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006593:Hexokinase; D005947:Glucose