Comparative study on the structure and stability of bovine seminal ribonuclease, its monomeric bis(S-carboxymethylated-31,32) derivative, and bovine pancreatic ribonuclease

Grandi, Claudio; D’Alessio, Giuseppe; Fontana, Angelo

doi:10.1021/bi00582a031

Cited by 18 publications

(13 citation statements)

References 56 publications

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“…It has already been reported (Sorrentino et al 2000) that in going from the monomer to the minor and to the major dimer, the CD signals become more positive at 242 nm, and less negative at 278 nm. This finding has been reported as being the evidence of a progressively higher exposure of the phenolic rings of tyrosine residues (Grandi et al 1979). Although the minor RNase A dimer is formed by the swapping of the N-terminal helixes (residues 1-15) of each subunit (Liu et al 1998), the structure of the major dimer is due to the domain swapping of the C-terminal ␤-strands (residues 116-124) of each monomer (Liu et al 2001).…”

Section: Circular Dichroism Analysesmentioning

confidence: 80%

“…9) are characterized by two dichroic bands, one negative, centered at 278 nm, the other positive, centered at 242 nm. These signals can reasonably be attributed to the phenolic rings of tyrosine residues (Simons and Blout 1968;Grandi et al 1979). It has already been reported (Sorrentino et al 2000) that in going from the monomer to the minor and to the major dimer, the CD signals become more positive at 242 nm, and less negative at 278 nm.…”

Section: Circular Dichroism Analysesmentioning

confidence: 96%

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Structural properties of trimers and tetramers of ribonuclease A

et al. 2001

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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...

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Section: Circular Dichroism Analysesmentioning

confidence: 80%

Section: Circular Dichroism Analysesmentioning

confidence: 96%

Structural properties of trimers and tetramers of ribonuclease A

et al. 2001

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“…Those of D‐I instead show a loss of secondary structure in the region around 210 nm, and a much higher positive band at 240 nm. The latter feature is suggestive of a major exposure in D‐I of the phenolic rings of tyrosine residue(s) [22].…”

Section: Resultsmentioning

confidence: 98%

The two dimeric forms of RNase A

Sorrentino

Barone

Bucci³

et al. 2000

FEBS Letters

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In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868^3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X-ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437^3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.z 2000 Federation of European Biochemical Societies.

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“…The linked M* monomer is much less active on RNA than the free M monomer [12, 171; it is regulable in catalysing the second, rate-limiting step of the reaction [lo], while M is not; has a conformation which can be clearly discriminated from that of M monomers, as has been shown with proteolytic probes [29], by circular dichroism and fluorescence studies, performed in the presence and in the absence of denaturants [30], and by 'H-NMR studies [31].…”

Section: Discussionmentioning

confidence: 99%

Reacquisition of quaternary structure by fully reduced and denatured seminal ribonuclease

Parente¹,

D’Alessio²

1985

European Journal of Biochemistry

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Air-regenerated monomers of bovine seminal ribonuclease have been found capable of reassociating into native dimers, whereas monomers refolded in the presence of a glutathione redox mixture do not reassociate into dimers [Smith, K. G., D'Alessio, G. and Schaffer, S. W. (1978) Biochemistry 17, 2633-26381. The crucial step in the process of regeneration of dimers is an isomerization step, which the newly refolded monomers undergo in order to reassociate into dimers. The two sulfhydryls at sequence positions 31 and 32 of the seminal RNAase chain, forming in the native dimer the intersubunit disulfides, have been found to have an important role in the refolding of the monomeric intermediates, as well as in the regeneration of dimers.The study of the acquisition of native structure of proteins has consisted mainly in the study of how denatured proteins regain their native conformations and functions through refolding of their polypeptide chains. Bovine pancreatic RNAase A has been one of the most favored model proteins in these studies, since the landmark paper by Sela et al. [l], who showed how the enzyme, after denaturation and reduction of all its disulfide bonds, could spontaneously reoxidize in the air regaining its native structure and function.Bovine seminal (BS) RNAase is strictly related to pancreatic RNAase A, both functionally and structurally. The two enzymes have identical bond specificity and mechanism of action [2]; the amino acid sequence of the BS-RNAase subunit is homologous to the sequence of RNAase A [3]; the three-dimensional structures of RNAase A and of BSRNAase subunit are largely identical [4], with identical pairing of the intrachain disulfides [5]. On the other hand, BSRNAase has peculiar features, which are not shared by the pancreatic enzyme: it has a dimeric structure [6] with the two subunits linked by interchain disulfides [7, 81 and is regulated in its catalytic activity [9, 101. Bovine seminal RNAase may thus be a precious tool in protein-folding studies as an oligomeric model protein, structurally and functionally related to RNAase A, to which it may be advantageously compared for the great body of information which is available in the literature on the folding of RNAase A (see [l 11 and references cited therein).Smith et al. [12] found that fully reduced and denatured BS-RNAase refolded in the presence of glutathione into active monomers, while only traces of dimeric protein were detected in the regeneration products. Earlier studies on air regeneration of reduced and denatured BS-RNAase [13, 141 had indicated that dimeric enzyme could be obtained as a product Enzyme. Ribonuclease (pancreatic and seminal) (EC 3.1.27.5).of refolding, although in low yields. The interest in clarifying this question has recently increased, as it has been found that monomeric BS-RNAase, although catalytically active, is not regulable in its catalytic action, unlike the native dimeric enzyme [lo].We report here the results of experiments on air regeneration of reduced and denatured BS-RNAase, in which...

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Comparative study on the structure and stability of bovine seminal ribonuclease, its monomeric bis(S-carboxymethylated-31,32) derivative, and bovine pancreatic ribonuclease

Abstract: The secondary and tertiary structure of dimeric seminal ribonuclease, its monomeric bis(S-carboxymethylated-31,32) derivative (MCM-sRNase), and bovine pancreatic

Cited by 18 publications

References 56 publications

Structural properties of trimers and tetramers of ribonuclease A

Structural properties of trimers and tetramers of ribonuclease A

The two dimeric forms of RNase A

Reacquisition of quaternary structure by fully reduced and denatured seminal ribonuclease

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