Agashe V R, Udgaonkar J B
National Centre for Biological Sciences, TIFR Centre, Bangalore, India.
Biochemistry. 1995 Mar 14;34(10):3286-99. doi: 10.1021/bi00010a019.
Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curves obtained at 14 different temperatures in the range 273-323 K have been used in conjunction with thermally-induced denaturation curves obtained in the presence of 15 different concentrations of GdnHCl to characterize the thermodynamics of cold and heat denaturation of barstar. The linear free energy model has been used to determine the excess changes in free energy, enthalpy, entropy, and heat capacity that occur on denaturation. The stability of barstar in water decreases as the temperature is decreased from 300 to 273 K. This decrease in stability is not accompanied by a change in structure as monitored by measurement of the mean residue ellipticities at both 222 and 275 nm. When GdnHCl is present at concentrations between 1.2 and 2.0 M, the decrease in stability with decrease in temperature is however so large that the protein undergoes cold denaturation. The structural transition accompanying the cold denaturation process has been monitored by measuring the mean residue ellipticity at 222 nm. The temperature dependence of the change in free energy, obtained in the presence of 10 different concentrations of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K. Values of the thermodynamic parameters governing the cold and the heart denaturation of barstar have been obtained with high precision by analysis of these bell-shaped stability curves. The change in heat capacity accompanying the unfolding reaction, delta Cp, has a value of 1460 +/- 70 cal mol-1 K-1 in water. The dependencies of the changes in enthalpy, entropy, free energy, and heat capacity on GdnHCl concentration have been analyzed on the basis of the linear free energy model. The changes in enthalpy (delta Hi) and entropy (delta Si), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the folded state, both have a negative value at low temperatures. With an increase in temperature delta Hi makes a less favorable contribution, while delta Si makes a more favorable contribution to the change in free energy (delta Gi) due to this interaction. The change in heat capacity (delta CPi) that occurs on preferential interaction of GdnHCl with the unfolded form has a value of only 53 +/- 36 cal mol-1 K-1 M-1. The data validate the linear free energy model that is commonly used to analyze protein stability.
在273 - 323 K范围内的14个不同温度下获得的盐酸胍（GdnHCl）等温诱导变性曲线，已与在15种不同浓度的GdnHCl存在下获得的热诱导变性曲线结合使用，以表征巴司他汀的冷热变性热力学。线性自由能模型已用于确定变性时自由能、焓、熵和热容的过量变化。随着温度从300 K降至273 K，巴司他汀在水中的稳定性降低。通过在222和275 nm处测量平均残基椭圆率监测，这种稳定性的降低并未伴随着结构的变化。然而，当GdnHCl的浓度在1.2至2.0 M之间时，随着温度降低稳定性的下降非常大，以至于蛋白质会发生冷变性。通过测量222 nm处的平均残基椭圆率监测了伴随冷变性过程的结构转变。在0.2 - 2.0 M范围内的10种不同浓度的GdnHCl存在下获得的自由能变化的温度依赖性表明，随着温度从300 K降低以及升高，稳定性都会下降。通过分析这些钟形稳定性曲线，高精度地获得了控制巴司他汀冷热变性的热力学参数值。在水中，伴随展开反应的热容变化ΔCp的值为1460±70 cal mol⁻¹ K⁻¹。基于线性自由能模型分析了焓、熵、自由能和热容变化对GdnHCl浓度的依赖性。相对于折叠态，GdnHCl优先与未折叠态结合时发生的焓变（ΔHi）和熵变（ΔSi）在低温下均为负值。随着温度升高，ΔHi对自由能变化（ΔGi）的贡献变得不太有利，而ΔSi的贡献变得更有利。GdnHCl与未折叠形式优先相互作用时发生的热容变化（ΔCPi）的值仅为53±36 cal mol⁻¹ K⁻¹ M⁻¹。这些数据验证了常用于分析蛋白质稳定性的线性自由能模型。
