title: Crowding agents and osmolytes provide insight into the formation and dissociation of RNase A oligomers.
abstract: RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A "C-dimer", the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.
authors.affiliation: Dipartimento di Chimica, Università degli Studi di Napoli Federico II, Via Cintia, 80126 Napoli, Italy.
authors.forename: Carmine
authors.initials: C
authors.lastname: Ercole
authors.forename: Jorge Pedro
authors.initials: JP
authors.lastname: López-Alonso
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: Delia
authors.initials: D
authors.lastname: Picone
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
mesh_terms: D000818:Animals; D002417:Cattle; D003911:Dextrans; D019281:Dimerization; D004795:Enzyme Stability; D005362:Ficoll; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D008744:Methylamines; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D003911:Dextrans; D008744:Methylamines; D021122:Protein Subunits; D005362:Ficoll; D012259:Ribonuclease, Pancreatic; C005855:trimethyloxamine