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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aleshin AE, Zeng C, Bourenkov GP, Bartunik HD, Fromm HJ, Honzatko RB (1998) The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose 6-phosphate. Structure 6:39–50
Arora KK, Filburn CR, Pedersen PL (1993) Structure/function relationships in hexokinase. Site-directed mutational analyses and characterization of overexpressed fragments implicate different functions for the N- and C-terminal halves of the enzyme. J Biol Chem 268:18259–18266
Blow DM, Steitz TA (1970) X-ray diffraction studies of enzymes. Annu Rev Biochem 39:63–100
Jeong J, Kim SK, Ahn J, Park K, Jeong EJ, Kim M, Chung BH (2006) Monitoring of conformational change in maltose binding protein using split green fluorescent protein. Biochem Biophys Res Commun 339:647–651
Kain SR, Ma JT (1999) Early detection of apoptosis with annexin V-enhanced green fluorescent protein. Methods Enzymol 302:38–43
Kamata K, Mitsuya M, Nishimura T, Eiki J, Nagata Y (2004) Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase. Structure 12:429–438
Mulichak AM, Wilson JE, Padmanabhan K, Garavito RM (1998) The structure of mammalian hexokinase-1. Nat Struct Biol 5:555–560
Ozawa T, Takeuchi TM, Kaihara A, Sato M, Umezawa Y (2001) Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein–protein interactions in bacteria: improved sensitivity and reduced screening time. Anal Chem 73:5866–5874
Pastorino JG, Hoek JB (2003) Hexokinase II: the integration of energy metabolism and control of apoptosis. Curr Med Chem 10:1535–1551
Rolland F, Sheen J (2005) Sugar sensing and signaling networks in plants. Biochem Soc Trans 33:269271
Rosano C, Sabini E, Rizzi M, Deriu D, Murshudov G, Bianchi M, Serafini G, Magnani M, Bolognesi M (1999) Binding of non-catalytic ATP to human hexokinase I highlights the structural components for enzyme-membrane association control. Structure 7:1427–1437
Stulke J, Hillen W (1999) Carbon catabolite repression in bacteria. Curr Opin Microbiol 2:195–201
Wilson JE (2003) Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol 206:2049–2057
Acknowledgements
This research was supported by grants from the Nano/Bio Science & Technology Program (MOST, Korea), and the KRIBB Initiative Research Program (KRIBB, Korea).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Jeong, EJ., Park, K., Joung, HA. et al. Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay. Biotechnol Lett 29, 797–802 (2007). https://doi.org/10.1007/s10529-007-9313-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-007-9313-x