Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3–5), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870–1874. 2. Libonati M. Int J Biochem 1969; 18: 407–417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311–327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437–3442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211–214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326–332.

Structural versatility of BS-RNase: Different oligomeric isomers formed through 3D domain swapping of N- and C-termini

GOTTE, Giovanni;
2011-01-01

Abstract

Bovine seminal RNase (BS RNase) is the only naturally dimeric member of the pancreatic-type RNase superfamily. The enzyme is a mixture of two isoforms: (i) M=M, dimeric through two antiparallel disulfides occurring between the two subunits; (ii) MxM (70% of the total), characterized by the swapping of the N-termini besides the mentioned disulfides (1). When dissolved in 40% acetic acid and subjected to lyophilisation, BS RNase forms a mixture of oligomeric aggregates (2), as does RNase A (3), the proto-type of the super-family. However, while the oligomers of the pancreatic variant have been extensively characterized (3–5), the multimers of BS RNase (6) are presently less known. To deepen their characterization we induced BS RNase self-association by using the same conditions used with RNase A, i.e. lyophilisation of enzyme solutions at low pH, or thermally-induced aggregation of concentrated protein solutions in various media. The multimers obtained were analyzed by SEC, cation-exchange chromatography, cross-linking, electrophoresis, proteolysis, and enzymatic assays. The main results obtained are: (a) BS-RNase forms several 3D domain-swapped conformers, in particular at least two tetramers; one is probably a totally N terminal-swapped isoform (4), while the other contains a C terminal swapping. (b) The C-swapped tetramer is less stable than the N-swapped one. (c) BS RNase multimers larger than tetramers appear to be a mixture of various isoforms, similarly to what occurs with RNase A (3). These multimers seem to contain, again, a Cterminus swapping. (4) The validity of the results reported have been strengthened by the studies performed with a K113N-BS RNase mutant. References: 1. Piccoli R., et al. Proc Natl Acad Sci USA 1992; 89: 1870–1874. 2. Libonati M. Int J Biochem 1969; 18: 407–417. 3. Libonati M. & Gotte G. Biochem J 2004; 380: 311–327. 4. Liu Y., et al. Proc Natl Acad Sci USA 1998; 95: 3437–3442. 5. Liu Y., et al. Nat Str Biol 2001; 8: 211–214. 6. Adinolfi S., et al. FEBS Lett 1996; 398: 326–332.
2011
bovine seminal ribonuclease; 3D domain swapping; ribonuclease oligomerization
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/786967
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? 0
social impact