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. At 29 degrees 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 degrees 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 monomer-dimer 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 conformation that completely displaces the equilibrium between nonswapped and swapped dimers to the latter one.
Rodríguez
Montserrat
M
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Spain.
Benito
Antoni
A
Ribó
Marc
M
Vilanova
Maria
M
D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry*; D012997:Solvents; D013329:Structure-Activity Relationship; D013696:Temperature
D012997:Solvents; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic