title: Rigidifying flexible sites: An approach to improve stability of chondroitinase ABC I.
abstract: The stability of chondroitin ABC lyase I (cABC I) at physiological temperature is one of the current obstacles to its clinical application. In this study, we used a protein engineering approach; rigidify flexible sites, to improve stability of cABC I. B-factor analysis showed a flexible loop at the N-terminal domain of cABC I which may be involved in its thermal instability and five residues in this region were replaced with proline. Thermal inactivation and thermal denaturation analysis revealed that Glu138Pro mutation increased half-life and Tm of enzyme, respectively. The Kmvalues of mutated enzymes were slightly increased compared to the wild type enzyme. The results of limited proteolysis indicated that Glu138Pro mutant was more resistant against trypsinolysis and this variant was less quenched in both acrylamide and KI quenching experiments. Moreover, intrinsic fluorescence intensity of Glu138Pro variant was increased and its ANS fluorescence intensity was decreased, whereas no considerable changes were observed in the far-UV CD spectra. The structural analyses indicated compactness of structure of Glu138Pro enzyme which can be related to moderately enhanced stability of this mutant. This study demonstrated that rigidifying flexible residues can be considered as a possible approach to increase the stability of the protein.
authors.affiliation: Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
authors.forename: Asma
authors.initials: A
authors.lastname: Kheirollahi
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.affiliation: Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran. Electronic address: golsetan@tums.ac.ir.
authors.forename: Abolfazl
authors.initials: A
authors.lastname: Golestani
mesh_terms: D019943:Amino Acid Substitution; D019765:Chondroitin ABC Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019277:Entropy; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D000072417:Protein Domains; D059748:Proteolysis; D044366:Transition Temperature
chemical_list: D019765:Chondroitin ABC Lyase
keywords: Chondroitinase ABC I; Flexibility and B-factor; Site directed mutagenesis; Stability