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Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Zelent B, Raimondo A, Barrett A, Buettger CW, Chen P, Gloyn AL, Matschinsky FM - Biochem. J. (2014)

Bottom Line: Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK.The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex.The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.

ABSTRACT
Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

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Structural stability and unfolding/refolding cycles of GKRP-WT and GKRP-P446LTF spectra for 0.3 μM GKRP in the presence or absence of 8 M urea at 20°C for GKRP-WT (A) and for GKRP-P446L (B). The horizontal arrows show the red and blue shifts of the spectra during denaturation and refolding at 4°C respectively. Vertical arrows show the TF spectra of the proteins in buffer normalized to 1. (C) TF spectra of WT-GK and (D) established instability mutant GK-S263P in the presence or absence of 8 M urea (λexc=295 nm).
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Figure 4: Structural stability and unfolding/refolding cycles of GKRP-WT and GKRP-P446LTF spectra for 0.3 μM GKRP in the presence or absence of 8 M urea at 20°C for GKRP-WT (A) and for GKRP-P446L (B). The horizontal arrows show the red and blue shifts of the spectra during denaturation and refolding at 4°C respectively. Vertical arrows show the TF spectra of the proteins in buffer normalized to 1. (C) TF spectra of WT-GK and (D) established instability mutant GK-S263P in the presence or absence of 8 M urea (λexc=295 nm).

Mentions: A measure of the structural stability of GKRP-WT and GKRP-P446L, as well as the ability of these two proteins to refold spontaneously upon denaturation, were critical for the biophysical studies described in the present paper [50,59]. We found that GKRP-WT is a relatively labile protein, as indicated by a ΔG(H2O) of 1.56 kcal/mol, and is comparable with GK-WT (1.63 kcal/mol). Both GKRP-WT and GK-WT refold readily upon denaturation with 8 M urea (Figure 4 and Supplementary Table S5 at http://www.biochemj.org/bj/459/bj4590551add.htm). Furthermore, GKRP-WT and GKRP-P446L displayed comparable ΔG(H2O) of denaturation (1.56 and 1.55 kcal/mol respectively) and unfolding/refolding patterns, suggesting that structural instability is unlikely to explain the metabolic effects of this variant protein. This conclusion is strengthened by results obtained by comparison with the proven instability mutants GK-M298K and GK-S263P [59] which clearly showed a lowering of their ΔG of denaturation and impaired refolding after urea denaturation (Figure 4 and Supplementary Table S5).


Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Zelent B, Raimondo A, Barrett A, Buettger CW, Chen P, Gloyn AL, Matschinsky FM - Biochem. J. (2014)

Structural stability and unfolding/refolding cycles of GKRP-WT and GKRP-P446LTF spectra for 0.3 μM GKRP in the presence or absence of 8 M urea at 20°C for GKRP-WT (A) and for GKRP-P446L (B). The horizontal arrows show the red and blue shifts of the spectra during denaturation and refolding at 4°C respectively. Vertical arrows show the TF spectra of the proteins in buffer normalized to 1. (C) TF spectra of WT-GK and (D) established instability mutant GK-S263P in the presence or absence of 8 M urea (λexc=295 nm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109836&req=5

Figure 4: Structural stability and unfolding/refolding cycles of GKRP-WT and GKRP-P446LTF spectra for 0.3 μM GKRP in the presence or absence of 8 M urea at 20°C for GKRP-WT (A) and for GKRP-P446L (B). The horizontal arrows show the red and blue shifts of the spectra during denaturation and refolding at 4°C respectively. Vertical arrows show the TF spectra of the proteins in buffer normalized to 1. (C) TF spectra of WT-GK and (D) established instability mutant GK-S263P in the presence or absence of 8 M urea (λexc=295 nm).
Mentions: A measure of the structural stability of GKRP-WT and GKRP-P446L, as well as the ability of these two proteins to refold spontaneously upon denaturation, were critical for the biophysical studies described in the present paper [50,59]. We found that GKRP-WT is a relatively labile protein, as indicated by a ΔG(H2O) of 1.56 kcal/mol, and is comparable with GK-WT (1.63 kcal/mol). Both GKRP-WT and GK-WT refold readily upon denaturation with 8 M urea (Figure 4 and Supplementary Table S5 at http://www.biochemj.org/bj/459/bj4590551add.htm). Furthermore, GKRP-WT and GKRP-P446L displayed comparable ΔG(H2O) of denaturation (1.56 and 1.55 kcal/mol respectively) and unfolding/refolding patterns, suggesting that structural instability is unlikely to explain the metabolic effects of this variant protein. This conclusion is strengthened by results obtained by comparison with the proven instability mutants GK-M298K and GK-S263P [59] which clearly showed a lowering of their ΔG of denaturation and impaired refolding after urea denaturation (Figure 4 and Supplementary Table S5).

Bottom Line: Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK.The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex.The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.

ABSTRACT
Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

Show MeSH
Related in: MedlinePlus