TAILIEUCHUNG - Báo cáo khoa học: Tertiary structure in 7.9 M guanidinium chloride ) the role of Glu53 and Asp287 in Pyrococcus furiosus endo-b-1,3-glucanase

The thermodynamic stability of family 16 endo-b-1,3-glucanase (EC ) from the hyperthermophilic archaeon Pyrococcus furiosusis decreased upon single (D287A, E53A) and double (E53A⁄D287A) muta-tion of Asp287 and Glu53. In accordance with the homology model predic-tion, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. | ỊFEBS Journal Tertiary structure in M guanidinium chloride - the role of Glu53 and Asp287 in Pyrococcus furiosus endo-p-1 3-glucanase Roberta Chiaraluce1 Rita Florio1 Sebastiana Angelaccio1 Giulio Gianese2 Johan F. T. van Lieshout3 John van der Oost3 and Valerio Consalvi1 1 Dipartimento di Scienze Biochimiche A. Rossi Fanelli Sapienza University di Roma Italy 2 Ylichron Srlc o ENEA Casaccia Research Center S. Maria di Galeria Italy 3 Laboratory of Microbiology Wageningen University the Netherlands Keywords double mutant glycoside hydrolases laminarinase protein stability thermodynamic stability Correspondence V. Consalvi Dipartimento di Scienze Biochimiche A. Rossi Fanelli Universita La Sapienza A. Moro 5 00185 Rome Italy Fax 39 06 4440062 Tel 39 06 49910939 E-mail consalvi@ Received 26 July 2007 revised 8 October 2007 accepted 10 October 2007 doi The thermodynamic stability of family 16 endo-b-1 3-glucanase EC from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single D287A E53A and double E53A D287A mutation of Asp287 and Glu53. In accordance with the homology model prediction both carboxylic acids are involved in the composition of a calcium binding site as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that in P. furiosus endo-b-1 3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in M guanidinium chloride. The persistence of tertiary structure in M guanidinium chloride a feature of the wild-type enzyme is observed also for the three variant proteins. The AGH2O values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximatelty 50 lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive with an energy of interaction of kJ-mol-1 suggesting a communication between the .

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