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1 Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain
2 Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200 D, B-3001 Leuven, Belgium
Reprint requests to: Maria Vilanova, Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain; e-mail: dbmvb{at}fc.udg.es; fax: 34-972-41-81-50.
FTIR spectroscopy was used to characterize and compare the temperature- and pressure-induced unfolding of ribonuclease A and a set of its variants engineered in a hydrophobic region of the C-terminal part of the molecule postulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two-state, reversible unfolding transition using both pressure and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I' band and the tyrosine band, or the maximum of the band assigned to the ß-sheet structure, corroborate the results of a previous study using fourth-derivative UV absorbance spectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure- and temperature-denaturated states of all the ribonucleases characterized retained some secondary structure. However, their spectral maxima were centered at different wavenumbers, which suggests that pressure- and temperature-denaturated states do not have the same structural characteristics. Nevertheless, there was close correlation between the pressure and temperature midpoint transition values for the whole series of protein variants, which indicated a common tendency of stability toward pressure and heat.
Keywords: Ribonuclease A; protein engineering; protein folding; pressure versus heat-induced unfolding; Fourier-transform infrared spectroscopy
Abbreviations: CD, circular dichroism • CFIS, chain folding initiation site • C-terminal, carboxy-terminal • DSC, differential scanning calorimetry • FTIR, Fourier transform infrared • HPLC, high-performance liquid chromatography • MES, 4-morpholine-ethanesulphonic acid • NMR, nuclear magnetic resonance • PCR, polymerase chain reaction • RNase A, bovine pancreatic ribonuclease A • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • UV, ultraviolet
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