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Mechanism of 3D domain swapping in bovine seminal ribonuclease.

The FEBS journal (2014-03-13)
Roberta Spadaccini, Carmine Ercole, Giuseppe Graziano, Rainer Wechselberger, Rolf Boelens, Delia Picone
ABSTRACT

3D domain swapping (3D-DS) is a complex protein aggregation process for which no unique mechanism exists. We report an analysis of 3D-DS in bovine seminal ribonuclease, a homodimeric protein whose subunits are linked by two disulfide bridges, based on NMR and biochemical studies. The presence of the covalent bonds between the subunits stabilizes the unswapped dimer, and allows distinct evaluation of the structural and dynamic effects of the swapping with respect to the dimerization process. In comparison with the monomeric subunit, which, in solution has a compact structure without any propensity for local unfolding, both swapped and unswapped dimers show increased flexibility. NMR analysis, together with urea denaturation and hydrogen–deuterium exchange data, indicates that the two dimers have increased conformational fluctuations. Furthermore, we found that the rate-limiting step of both the swapping and unswapping pathways is the detachment of the N-terminal helices from the monomers. These results suggest a new general mechanism in which a dimeric intermediate could facilitate 3D-DS in globular proteins.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ribonuclease B from bovine pancreas, BioReagent, ≥50 Kunitz units/mg protein, ≥80% (SDS-PAGE)
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type I-AS, 50-100 Kunitz units/mg protein
Sigma-Aldrich
Ribonuclease A from bovine pancreas, for molecular biology, ≥70 Kunitz units/mg protein, lyophilized
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type XII-A, ≥90% (SDS-PAGE), 75-125 Kunitz units/mg protein
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type I-A, powder, ≥60% RNase A basis (SDS-PAGE), ≥50 Kunitz units/mg protein
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type X-A, ≥90% (SDS-PAGE), ≥70 Kunitz units/mg protein
Sigma-Aldrich
Ribonuclease A from bovine pancreas, (Solution of 50% glycerol, 10mM Tris-HCL pH 8.0)
Sigma-Aldrich
Ribonuclease A from bovine pancreas, 4×cryst., ~70 U/mg (acc. to Kunitz)
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type III-A, ≥85% RNase A basis (SDS-PAGE), 85-140 Kunitz units/mg protein
Sigma-Aldrich
Ribonuclease A from bovine pancreas, Type II-A, ≥60% (SDS-PAGE), >= 60 Kunitz units/mg protein