title: Pressure versus heat-induced unfolding of ribonuclease A: the case of hydrophobic interactions within a chain-folding initiation site.
abstract: To investigate the characteristics of the postulated carboxy terminal chain-folding initiation site in bovine pancreatic ribonuclease A (RNase A) (residues 106-118), important in the early stages of the folding pathway, we have engineered by site-directed mutagenesis a set of 14 predominantly conservative hydrophobic variants of the protein. The stability of each variant has been compared by pressure and temperature-induced unfolding, monitored by fourth derivative UV absorbance spectroscopy. Apparently simple two-state, reversible unfolding transitions are observed, suggesting that the disruption of tertiary structure of each protein at high pressure or temperature is strongly cooperative. Within the limits of the technique, we are unable to detect significant differences between the two processes of denaturation. Both steady-state kinetic parameters for the enzyme reaction and UV CD spectra of each RNase A variant indicate that truncation of hydrophobic side chains in this region has, in general, little or no effect on the native structure and function of the enzyme. Furthermore, the decreases in free energy of unfolding upon pressure and thermal denaturation of all the variants, particularly those modified at residues 106 and 108, suggest that the hydrophobic residues and side chain packing interactions of this region play an important role in maintaining the conformational stability of RNase A. We also demonstrate the potential of Tyr115 replacement by Trp as a non-destabilizing fluorescence probe of conformational changes local to the region.
authors.affiliation: Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi. E-17071 Girona, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Torrent
authors.forename: J P
authors.initials: JP
authors.lastname: Connelly
authors.forename: M G
authors.initials: MG
authors.lastname: Coll
authors.forename: M
authors.initials: M
authors.lastname: Ribó
authors.forename: R
authors.initials: R
authors.lastname: Lange
authors.forename: M
authors.initials: M
authors.lastname: Vilanova
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D004795:Enzyme Stability; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011312:Pressure; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D012259:Ribonuclease, Pancreatic