TAILIEUCHUNG - Báo cáo khoa học: Characterization of the dimerization process of a domain-swapped dimeric variant of human pancreatic ribonuclease

It has been previously reported that the structure of a human pancreatic ribonuclease variant, namely PM8, constitutes a dimer by the exchange of an N-terminal domain, although in an aqueous solution it is found mainly as a monomer. First, we investigated the solution conditions that favour the dimerization of this variant. | iFEBS Journal Characterization of the dimerization process of a domain-swapped dimeric variant of human pancreatic ribonuclease Montserrat Rodríguez Antoni Benito Marc Ribo and Maria Vilanova Laboratori d Enginyeria de Protei nes Departament de Biologia Facultat de Ciencies Universitat de Girona Spain Keywords 3D domain swapping dimerization mechanism human pancreatic ribonuclease Correspondence M. Vilanova Laboratori d Enginyeria de Protei nes Departament de Biologia Facultat de Ciencies Universitat de Girona Campus de Montilivi s n 17071 Girona Spain Fax 34 972 418150 Tel 34 972 418173 E-mail Received 12 December 2005 revised 10 January 2006 accepted 16 January 2006 doi It has been previously reported that the structure of a human pancreatic ribonuclease variant namely PM8 constitutes a dimer by the exchange of an N-terminal domain although in an aqueous solution it is found mainly as a monomer. First we investigated the solution conditions that favour the dimerization of this variant. At 29 C in a 20 v v ethanol buffer a significant fraction of the protein is found in dimeric form without the appearance of higher oligomers. This dimer was isolated by size-exclusion chromatography and the dimerization process was studied. The dissociation constant of this dimeric form is 5 mM at 29 C. Analysis of the dependence of the dimerization process on the temperature shows that unlike bovine pancreatic ribonuclease a decrease in the temperature shifts the monomerdimer equilibrium to the latter form. We also show that a previous dissociation of the exchangeable domain from the main protein body does not take place before the dimerization process. Our results suggest a model for the dimerization of PM8 that is different to that postulated for the dimerization of the homologous bovine pancreatic ribonuclease. In this model an open interface is formed first and then intersubunit interactions stabilize the hinge loop in a .

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