Thermodynamics of Core Hydrophobicity and Packing in the Hyperthermophile Proteins Sac7d and Sso7d
详细信息 查看全文
- 作者：Andrew T. Clark ; Bradford S. McCrary ; Stephen P. Edmondson ; and John W. Shriver
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 16, 2004
- 年：2004
- 卷：43
- 期：10
- 页码：2840 - 2853
- 全文大小：489K
- 年卷期：v.43,no.10(March 16, 2004)
- ISSN：1520-4995
文摘
The influence of core hydrophobicity and packing on the structure and stability of thehyperthermophile proteins Sac7d and Sso7d have been studied by calorimetry, circular dichroism, andNMR. Valine 30 is positioned in Sac7d to allow a cavity-filling Val 
Ile substitution which occursnaturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has beencharacterized and compared to the reciprocal Ile Val mutation in Sso7d. A detailed analysis of thestability of the proteins was obtained by globally fitting the variation of DSC parameters and circulardichroism intensities as a function of temperature (0-100 
C), salt (0-0.3 M), and pH (0-8). A globalanalysis over such a range of conditions permitted an unusually precise measure of the thermodynamicparameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from thelinked effects of protonation and chloride binding associated with acid-induced folding. The results indicatedifferences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysisof DSC data alone using conventional methods. The sign and magnitude of the changes in 
G, H, TS,and CP of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent withdifferences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions.The benefit of increased hydrophobicity of the core increased with temperature, with maximal effectaround 116 C. Increased hydrophobicity of the core achieved not only an increase in the free energy ofunfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.
Ile substitution which occursnaturally in the homologous more thermostable Sso7d. The cavity-filling mutation in Sac7d has beencharacterized and compared to the reciprocal Ile Val mutation in Sso7d. A detailed analysis of thestability of the proteins was obtained by globally fitting the variation of DSC parameters and circulardichroism intensities as a function of temperature (0-100 C), salt (0-0.3 M), and pH (0-8). A globalanalysis over such a range of conditions permitted an unusually precise measure of the thermodynamicparameters, as well as the separation of the thermodynamics of the intrinsic unfolding reaction from thelinked effects of protonation and chloride binding associated with acid-induced folding. The results indicatedifferences in the energetics of unfolding Sac7d and Sso7d that would not be apparent from an analysisof DSC data alone using conventional methods. The sign and magnitude of the changes in G, H, TS,and CP of unfolding resulting from core Ile/Val substitutions in the two proteins were consistent withdifferences in hydrophobicity of Val and Ile and negligible changes in packing (van der Waals) interactions.The benefit of increased hydrophobicity of the core increased with temperature, with maximal effectaround 116 C. Increased hydrophobicity of the core achieved not only an increase in the free energy ofunfolding, but also a lateral shift of the temperature of maximal stability to higher temperature.