Mapping the stability clusters in bovine pancreatic ribonuclease A.
In the present work, we have thermodynamically characterized the thermally induced unfolding of 20 variants of bovine pancreatic ribonuclease A (RNase A) to experimentally describe the residues and the regions that are critical for the stability of the enzyme. The achieved results, complemented with previous studies by our group, allowed us to define the significance of the two hydrophobic nuclei present in the RNase A structure, as well as the contribution of the participating residues within each nucleus, to the global enzyme stability. We propose a structural model for the major and the minor hydrophobic nuclei of RNase A. The major nucleus is composite and located in the cavity delimited by alpha-helices 1 and 3, and the beta-sheet that is formed by strands 2, 3, 5, and 6. It consists of a central tight packed part constituted by residues Phe8, Met13, Val54, Val57, Ile106, Val108, and Phe120. This central part is surrounded by a layer formed by residues Val63, Tyr73, Met79, Ile107, Val116, and Val118. The minor nucleus, although less complex, is also constituted by a tight packing that involves the side chains of residues Tyr25, Met29, Met30, Leu35, Phe46, and Tyr97, which fill the cavity that originates the beta-sheet formed by beta-strands 1, 4, and 5 together with alpha-helix2.
Departament de Biologia, Universitat de Girona, Campus de Montilivi s/n 17071 Girona, Spain.
Roger
R
Vilà
Antoni
A
Benito
Marc
M
Ribó
Maria
M
Vilanova
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004795:Enzyme Stability; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
D000596:Amino Acids; D007527:Isoenzymes; D012259:Ribonuclease, Pancreatic