Structural versatility of bovine ribonuclease A

Abstract

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.

Structural properties of trimers and tetramers of ribonuclease A

“…Although in fact the minor and major dimeric conformers are always in the ratio of at least 1:3-1:4, so that the formation of the major RNase A dimer appears to be definitely favored if compared to that of the minor dimer, no such great difference can be observed in the relative amounts of the two trimeric or tetrameric conformers. The relative proportions of monomeric RNase A and its various aggregates (average values calculated from 15 experiments; see also Gotte et al 1999) are, in fact, the fol- Fig. 10.…”

“…Absorbances at 260 nm were determined with a Beckman DU 650 spectrophotometer, equipped with a thermostatically controlled water bath, after maintaining the nucleic acid-protein mixture at the chosen temperature for 2.5 min (i.e., after hyperchromicity reached a stable value). residues in the enzyme protein, through its ability to destabilize the nucleic acid secondary structure; and (2) therefore, explains why the more basic conformers of each RNase A aggregate degrade double-stranded RNA more efficiently than the less basic conformers (Gotte et al 1999 The integrity of the structure of the RNase A aggregates is a crucial point for the validity of the experiment shown in Figure 3. From the experimental results presented in Figure 4, in which the more basic dimeric (D 2 ), trimeric (T 2 ), and tetrameric (TT 2 ) conformers in the absence of the nucleic acid were held at the temperatures indicated for the same times (2.5 min) where they were maintained when their helix-unwinding activity was examined ( Fig.…”

“…Properties of trimeric and tetrameric RNase A www.proteinscience.org 2025 (Gotte et al 1999), was collected and used for all following procedures.…”

“…About 35 mg protein (in 2 mL) were applied and eluted at room temperature with a linear gradient of sodium phosphate buffer, pH 6.7 (0.09-0.18 M, 120 min), at a flow rate of 1.2 mL/min. Under these experimental conditions the various oligomers elute with a pattern identical to that already described (Gotte et al 1999). The peaks of interest were finally collected and used immediately or kept frozen until use.…”

“…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.…”