3D Domain Swapping Provides a Minor Alternative Refolding Pathway for Ribonuclease A
Abstract:The C-terminal β-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal β-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C terminal β-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit β-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.
Keywords: 3D domain swapping; 3D domain-swapped oligomers; C-dimer; GdmCl; Hofmeister stabilizer; NaPi buffer; RNase A; amyloid formation; amyloidogenesis; chromatography; hydrogen deuterium exchange; proline isomerization; protein folding
Document Type: Research Article
Publication date: May 1, 2011
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