MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer FEBS Lett. (0014-5793) 2013 imex curation Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro bn-page blue native page bn-page weight by staining molecular weight estimation by staining weight by staining Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it Accepted 2013-OCT-07 by LPERFETTO 28-Oct_2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer FEBS Lett. (0014-5793) 2013 imex curation Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro x-ray diffraction x-ray crystallography X-ray X-ray x-ray diffraction predetermined predetermined participant predetermined Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it Accepted 2013-OCT-07 by LPERFETTO 28-Oct_2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer The multiple forms of Bovine Seminal Ribonuclease: structure and stability of a C-terminal swapped dimer FEBS Lett. (0014-5793) 2013 imex curation Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Sica F., Pica A., Merlino A., Krauss I.R., Ercole C., Picone D. delia.picone@unina.it Accepted 2013-OCT-07 by LPERFETTO 28-Oct_2013: Contacted by IntAct-Help. FEBS Lett. (0014-5793) 2013 Only protein-protein interactions imex curation rns_bovin Seminal ribonuclease SRN Ribonuclease BS-1 protein protein bovin Bos taurus (Bovine) Bovine MALKSLVVLPLLVLVLLLVRVQPSLGKESAAAKFERQHMDSGNSPSSSSNYCNLMMCCRKMTQGKCKPVNTFVHESLADVKAVCSQKKVTCKNGQTNCYQSKSTMRITDCRETGSSKYPNCAYKTTQVEKHIIVACGGKPSVPVHFDASV mint F7A05C930FB83A83 srn-1 3 BS-RNase 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified direct interaction direct interaction f1 line). Peak integration revealed that more than 60% of the protein is eluted as monomer, whilst the remaining part corresponds to aggregated forms of different size, although the relative amount of each aggregate varied slightly with the elution buffer. From the elution volume it is possible to calculate that the peak indicated as TrART corresponds to the size of a trimer, and the one indicated as DART to the size of a dimer. The trimer and dimer amounts are equivalent roughly to the 10% and 20-25% of the total protein amount respectively. A similar experiment was performed on the STAA-mBS mutant, obtaining the chromatographic profile also reported in Figure 1 (solid line), which closely resembles the one obtained with the parent (wild-type) protein, except for the slightly higher amount of aggregates. Very similar results were obtained with other BS-RNase mutants, in which residues having key roles in the swapping phenomenon were substituted with the corresponding ones of RNase A and which have a different behaviour with respect to the swapping of the N5 terminal ends, i.e.R80S-STAA-mBS [27] and PALQ-mBS [28]. The elution profile of PALQ-mBS is also shown in Fig. 1 as an example (dotted line). srn-1 3 BS-RNase 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified direct interaction direct interaction f1 line). Peak integration revealed that more than 60% of the protein is eluted as monomer, whilst the remaining part corresponds to aggregated forms of different size, although the relative amount of each aggregate varied slightly with the elution buffer. From the elution volume it is possible to calculate that the peak indicated as TrART corresponds to the size of a trimer, and the one indicated as DART to the size of a dimer. The trimer and dimer amounts are equivalent roughly to the 10% and 20-25% of the total protein amount respectively. A similar experiment was performed on the STAA-mBS mutant, obtaining the chromatographic profile also reported in Figure 1 (solid line), which closely resembles the one obtained with the parent (wild-type) protein, except for the slightly higher amount of aggregates. Very similar results were obtained with other BS-RNase mutants, in which residues having key roles in the swapping phenomenon were substituted with the corresponding ones of RNase A and which have a different behaviour with respect to the swapping of the N5 terminal ends, i.e.R80S-STAA-mBS [27] and PALQ-mBS [28]. The elution profile of PALQ-mBS is also shown in Fig. 1 as an example (dotted line). srn-1 1 BS-RNase 4 weight by staining molecular weight estimation by staining weight by staining weight by staining molecular weight estimation by staining weight by staining unspecified role unspecified role unspecified role unspecified role purified purified direct interaction direct interaction f2 The analysis of the gel indicated clearly that BS-RNase and NCD-BS (lanes 3 and 4) are characterized by a single band. On the contrary, both the artificial dimers (lanes 5 and 6) showed the presence of a significant amount of the corresponding monomeric forms (lanes 1 and 2), which necessarily had to be produced upon dissociation of the dimeric species after the gel-filtration and even while running the gel. Interestingly, the comparison of lanes 3 and 4 with 5 and 6 indicates that the artificial dimers migrate on the gel with a mobility very close to that of NCD-BS, thus suggesting the presence of a single dimeric form even in the samples prepared by acetic acid lyophilisation, in contrast with the results obtained for RNase A. It is indeed well known that the two dimers formed when RNase A is subjected to the same treatment can be separated by cathodic gel-electrophoresis [36]. srn-1 2 BS-RNase 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role unspecified role unspecified role purified purified direct interaction direct interaction f3 f4 t1