Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot |
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Authors: | Mahdieh Nazari-Robati Khosro Khajeh Mahdi Aminian Nasrin Mollania Abolfazl Golestani |
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Affiliation: | 1. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran;2. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran;3. Department of Biology, Faculty of Basic Sciences, Hakim Sabzevari University, Sabzevar, Iran |
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Abstract: | The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein. |
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Keywords: | cABC I, Chondroitinase ABC I C6S, Chondroitin-6-sulfate C4S, Chondroitin-4-sulfate DS, Dermatan sulfate GAGs, Glycosaminoglycans CSPG, Chondroitin sulfate proteoglycan SCI, Spinal cord injury CD, Circular dichroism PDB, Protein Data Bank |
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