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Oligomerization of ribonuclease A under reducing conditions
No full textBy lyophilization from 40% acetic acid solutions, bovine ribonuclease A forms well characterized, three-dimensional domain-swapped oligomers: dimers, trimers, tetramers, and higher order multimers. Each oligomeric species consists of at least two conformers. Identical oligomers also form by thermally-inducing the oligomerization of highly concentrated RNase A dissolved in fluids endowed with various denaturing power. Now, our question is: which might the influence of a reducing agent be on RNase A oligomerization, i.e., of conditions that decrease the stability of the protein and increase the mobility of its swapping domains? To address this question, we carried out experiments of RNase A oligomerization in the presence of increasing concentrations of dithiothreitol (DTT) under the two experimental conditions mentioned above. Results indicate that RNase A oligomers similar to those previously known form anyhow, but with a change of their relative proportions. The amounts of dimers and trimers decrease by increasing the concentration of DTT, while the yields of two tetramers remarkably increase. Moreover, in the presence of DTT RNase A forms labile and probably unstructured aggregates that can possibly drive the protein towards precipitation when the reducing agent's concentration increases. Taken together, these results point out once again (i) the important role of the 3D domain swapping mechanism in protein oligomerization, and (ii) the importance of the native structure of RNase A (and of proteins in general) in preventing an uncontrolled aggregation and precipitation in a reducing and highly crowded environment like that existing in a living cell. (c) 2007 Elsevier B.V. All rights reserved
Oligomerization of ribonuclease A. Two novel three-dimensional domain-swapped tetramers
No full textBy lyophilization from 40% acetic acid solutions, bovine
ribonuclease A forms several types of three-dimensional
domain-swapped oligomers: dimers, trimers, tetramers,
and higher order multimers. Each oligomeric
species comprehends at least two conformers: one less
basic and one more basic. The structures of the two
dimers and one trimer have been solved. Plausible models
have been proposed for the other oligomers. Among
them, all chromatographic patterns show the constant
presence of minority species, and we focused our attention
on two of them. The first oligomer (named X)
elutes between the two trimeric conformers; the second
(named Y) elutes as a shoulder in the ascending limb of
the more basic trimer. After purification with cationexchange
chromatography, on the basis of (a) gel filtration
analyses, (b) gel electrophoreses under nondenaturing
conditions, (c) SDS-PAGE, (d) cross-linking experiments
with divinylsulfone and 1,5-difluoro 2,4-
dinitrobenzene, (e) enzymatic activity assays, (f)
identification of the products of their spontaneous dissociation,
and (g) controlled proteolysis with subtilisin,
we propose that the X and Y oligomeric species contain
two novel three-dimensional domain-swapped tetrameric
conformers of RNase A, differing from each
other as well as from the two tetramers already identified.
For the two novel tetramers we showed tentative
structural models. XTT could be a circular NCNCtetramer;
YTT could be a propeller-like C-trimer with an
attached N-swapping monomer (NCCCTT), identical to a
model proposed by Liu and Eisenberg (Liu, Y., and
Eisenberg, D. (2002) Protein Sci. 11, 1285–1299)
Oligomerization of ribonuclease A. Structural and functional features of its multimers
No full textBovine 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 (threedimensional)
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 RNaseA,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
[Endonucleolytic mechanism of degradation of double-helix RNA by ribonuclease BS-1 and by ribonuclease A aggregates]
No full textEndonucleolytic mechanism of degradation of double-helix RNA by ribonuclease BS-1 and by ribonuclease A aggregate
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