Oligomerization of ribonuclease A: two novel three-dimensional domain-swapped tetramers.
By 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 cation-exchange 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. X(TT) could be a circular NCNC-tetramer; Y(TT) could be a propeller-like C-trimer with an attached N-swapping monomer (NCCC(TT)), identical to a model proposed by Liu and Eisenberg (Liu, Y., and Eisenberg, D. (2002) Protein Sci. 11, 1285-1299).
Gotte
Giovanni
G
Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, Strada Le Grazie 8, I-37134 Verona, Italy.
Libonati
Massimo
M
D000818:Animals; D002412:Cations; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004139:Dinitrofluorobenzene / Q000031:analogs & derivatives* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D008958:Models, Molecular; D010179:Pancreas / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D020860:Subtilisin / Q000737:chemistry; D013440:Sulfides / Q000737:chemistry; D013696:Temperature; D013997:Time Factors
D002412:Cations; D003432:Cross-Linking Reagents; D013440:Sulfides; C013523:ethylene sulfide; D004139:Dinitrofluorobenzene; D012259:Ribonuclease, Pancreatic; D020860:Subtilisin; C002041:1,5-difluoro-2,4-dinitrobenzene