Three-dimensional domain-swapped oligomers of ribonuclease A: Identification of a fifth tetramer, pentamers and hexamers, and detection of trace heptameric, octameric and nonameric species

摘要

By lyophilization from 40% acetic acid solutions, bovine pancreatic Ribonuclease A forms three-dimensional domain-swapped dimers, trimers, and tetramers that can be separated by cation-exchange chromatography. Each oligomeric species consists of at least two conformers, one less basic, one more basic. The structures of the two dimers and one trimer have been solved. Plausible models have been proposed for a second RNase A trimer and four tetramers. This work is focused on the characterization of the largest oligomers which compose small peaks that have always appeared in chromatograms of RNase A. These higher order oligomers were collected by repeated cation-exchange chromatographies. On the basis of (a) gel filtrations through analytical Superdex 75 and 200; (b) gel electrophoreses under non-denaturing conditions, (c) cross-linking with divynilsulfone followed by analyses with SDS-PAGE and mass spectrometry, (d) enzymatic activity assays, and (e) analyses of the products of spontaneous dissociation of the oligomers, we could identify three-dimensional domain-swapped pentamers and hexamers, and one additional ietrameric conformer. For the latter we propose a cyclic model (C-TT). Moreover, we advance a linear model (NCNCp) for one pentamer, and three possible cyclic models (with a C-trimer as the main component) for one hexamer. The experimental evidence also indicates the existence of heptameric, octameric and nonameric species. (c) 2005 Elsevier B.V. All rights reserved.