Torrent J, Rubens P, Ribó M, Heremans K, Vilanova M
Laboratori d'Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, E-17071 Girona, Spain.
Protein Sci. 2001 Apr;10(4):725-34. doi: 10.1110/ps.43001.
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 beta-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.
傅里叶变换红外光谱（FTIR）被用于表征和比较核糖核酸酶A及其在假定为CFIS的分子C末端疏水区域设计的一组变体在温度和压力诱导下的去折叠情况。结果表明，对于所有研究的核糖核酸酶，使用压力和温度均可诱导出协同、两态、可逆的去折叠转变。通过酰胺I'带和酪氨酸带最大值位置的变化，或β-折叠结构相关带的最大值来监测不同位点以及同一位点不同变体之间的相对稳定性，这证实了先前使用四阶导数紫外吸收光谱研究的结果。此外，108位的变体对核糖核酸酶的结构和稳定性最为关键。V108G变体似乎比其他变体具有更大的构象灵活性。所有表征的核糖核酸酶的压力和温度变性状态都保留了一些二级结构。然而，它们的光谱最大值集中在不同的波数，这表明压力和温度变性状态不具有相同的结构特征。尽管如此，整个系列蛋白质变体的压力和温度中点转变值之间存在密切相关性，这表明在压力和热稳定性方面存在共同趋势。
