title: NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping.
abstract: Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.
authors.affiliation: Instituto de Química Física Rocasolano, C.S.I.C., Serrano 119, E-28006 Madrid, Spain.
authors.forename: Jorge Pedro
authors.initials: JP
authors.lastname: López-Alonso
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.forename: Josep
authors.initials: J
authors.lastname: Font
authors.forename: Marc
authors.initials: M
authors.lastname: Ribó
authors.forename: Maria
authors.initials: M
authors.lastname: Vilanova
authors.forename: María Angeles
authors.initials: MA
authors.lastname: Jiménez
authors.forename: Jorge
authors.initials: J
authors.lastname: Santoro
authors.forename: Carlos
authors.initials: C
authors.lastname: González
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
mesh_terms: D019342:Acetic Acid / Q000737:chemistry; D000818:Animals; D002417:Cattle; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D012259:Ribonuclease, Pancreatic; D019342:Acetic Acid