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Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes


Affiliations
1 Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India
 

Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys23-Cys63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.

Keywords

Protein Unfolding, Stem Bromelain, Urea Gradient, Zymography.
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  • Application of Transverse Urea Gradient Zymography for Structural and Functional Characterization of Proteolytic Enzymes

Abstract Views: 289  |  PDF Views: 89

Authors

Kanika Sharma
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India
Debasish Bhattacharyya
Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, Jadavpur, Kolkata 700 0032, India

Abstract


Inactivation of an enzyme as it begins to unfold, along with the conformational perturbations which follow, can provide an insight into dynamics of the unfolding pathway. Urea gradient electrophoresis combined with zymography is a sensitive technique which provides a continuous visual profile of a proteolytic enzyme undergoing denaturation and inactivation simultaneously. Trypsin has been used as a reference protease to validate and standardize the method by correlating inactivation profile generated in zymography with a solution state assay. Stem bromelain, a cysteine endopeptidase was used as a case study to evaluate this methodology. The method highlighted the effect rendered by the substrate on the stability of the proteolytic domain of the enzyme, as it undergoes urea-induced unfolding. Transverse urea gradient zymography combined with molecular modelling of stem bromelain, where the disulphide bonds have been reduced, indicated that the evolutionary retention of Cys23-Cys63 could be attributed to localized stabilization imparted by this bond to the catalytic site. This method encompasses various dimensions to extend the understanding of structure-function relationship in denaturant-induced unfolding pathways of proteases.

Keywords


Protein Unfolding, Stem Bromelain, Urea Gradient, Zymography.

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DOI: https://doi.org/10.18520/cs%2Fv111%2Fi8%2F1340-1348