Lyophilization of bovine ribonuclease A (RNase A; Sigma, type XII-A) from 40% acetic acid solutions leads to the formation of < 14 aggregated species that can be separated by ion-exchange chromatography. Several aggregates were identified, including two variously deamidated dimeric subspecies, two distinct trimeric and two distinct tetrameric RNase A conformers, besides the two forms of dimer characterized previously [Gotte, G. & Libonati, M. (1998) Two different forms of aggregated dimers of ribonuclease A. Biochim. Biophys. Acta 1386, 106±112]. We also have possible evidence for the existence of two forms of pentameric RNase A. The two forms of trimers and tetramers are characterized by: (a) slightly different gel filtration patterns; (b) different retention times in ion-exchange chromatography; and (c) different mobilities in cathodic gel electrophoresis under nondenaturing conditions. Therefore, they appear to have distinct structural organizations responsible for a different availability of their positively charged amino acid residues. All RNase A oligomers, in particular the two distinct trimeric and tetrameric conformers, degrade poly(A)´poly(U), viral double-stranded RNA and polyadenylate with a catalytic efficiency that is in general higher for the more basic species. On the contrary, the activity of the RNase A oligomers, from dimer to pentamer, on yeast RNA and poly(C) (Kunitz assay) is lower than that of monomeric RNase A.

Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.

Bovine pancreatic ribonuclease (RNase A) forms two types of dimers (a major and a minor component) upon concentration in mild acid. These two dimers exhibit different biophysical and biochemical properties. Earlier we reported that the minor dimer forms by swapping its N-terminal alpha-helix with that of an identical molecule. Here we find that the major dimer forms by swapping its C-terminal beta-strand, thus revealing the first example of three-dimensional (3D) domain swapping taking place in different parts of the same protein. This feature permits RNase A to form tightly bonded higher oligomers. The hinge loop of the major dimer, connecting the swapped beta-strand to the protein core, resembles a short segment of the polar zipper proposed by Perutz and suggests a model for aggregate formation by 3D domain swapping with a polar zipper.

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Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) · poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) · poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.Keywords: RNase A oligomers; trimers and tetramers of RNase A; properties of trimeric and tetrameric RNase A; RNase A aggregates higher than dimersThe study of the manner in which proteins aggregate in vitro can help in understanding the process of protein aggregation in vivo, and, therefore, also the origin of pathologic proteins responsible for several severe diseases.Although it has recently been reported that native ribonuclease A can dimerize at neutral pH (Park and Raines 2000), it is known that by lyophilization from 30-50% acetic acid solutions RNase A gives rise to oligomers (Crestfield et al. 1962) ranging from dimers to pentamers and possibly higher aggregates, each oligomeric species existing in the form of at least two conformational isomers (Gotte et al. 1999).Dimeric RNase A obtained by lyophilization consists of a minor and a major component that are in the ratio of about 1:3-1:4. Their crystal structures have been determined (Liu et al. 1998(Liu et al. , 2001, and the two dimeric conformers are 3D domain-swapping dimers formed by the exchange of the N-terminal ␣-helix of each monomeric subunit in the case of the minor dimer, of the C-terminal ␤-strand, instead, for the major RNase A dimer. This unique behavior of RNase A, in the dimeric aggregates of which the two known mechanisms of protein aggregation by domain swapping coexist, expands the range of possibi...