Backmann J, Schäfer G, Wyns L, Bönisch H
Dienst Ultrastruktuur, Vrije Universiteit Brussel, Paardenstraat 65, Sint-Genesius-Rode, B-1640, Belgium.
J Mol Biol. 1998 Dec 4;284(3):817-33. doi: 10.1006/jmbi.1998.2216.
The thermal stability of adenylate kinase from the thermoacidophilic archaeon Sulfolobus acidocaldarius was characterized comprehensively using denaturant-induced unfolding, differential scanning calorimetry, circular dichroism spectroscopy, and enzymological inactivation studies. The thermally induced unfolding of the protein is irreversible due to aggregation, whereas the unfolding induced by guanidinium chloride is reversible. The protein is known to be a homotrimer in its native state and we established that it unfolds upon dissociation in the case of denaturant unfolding. We measured the thermodynamic stability of the protein in a temperature range from 5 to 70 degrees C using denaturant unfolding. The protein has a maximum of stability (intrinsic free energy) of 31 kcal/mol-trimer (130 kJ/mol-trimer) at 32 degrees C (based on the linear extrapolation model). The heat capacity change upon unfolding DeltaCp and the m-value were considered to be constant in this temperature range and calculated to be 2.86 kcal/mol-trimer (11.9 kJ/mol-trimer) and 5.67 kcal/mol-trimer M (23.7 kJ/mol-trimer M), respectively. The influence of trimerization on thermodynamic stability was investigated. The several interrelated aspects of thermal stability such as unfolding kinetics, the temperature-dependence of the free energy, and the concentration and temperature-dependencies of the fraction of denatured protein are described quantitatively. The properties of the Gibbs-Helmholtz function of the adenylate kinase from S. acidocaldarius, in particular, and of oligomeric proteins, in general terms, are discussed and compared with the properties of the analogous function for monomeric proteins. Moreover, we discuss methodological aspects: we obtained the analytical expression of the denaturant-unfolding isotherm for homotrimeric proteins; we include a formula Appendix containing the derivations of the expressions used.
利用变性剂诱导的去折叠、差示扫描量热法、圆二色光谱法和酶学失活研究，全面表征了嗜热嗜酸古菌嗜酸热硫化叶菌腺苷酸激酶的热稳定性。由于聚集作用，该蛋白的热诱导去折叠是不可逆的，而氯化胍诱导的去折叠是可逆的。已知该蛋白在其天然状态下是同三聚体，并且我们确定在变性剂去折叠的情况下，它在解离时会去折叠。我们使用变性剂去折叠测量了该蛋白在5至70摄氏度温度范围内的热力学稳定性。基于线性外推模型，该蛋白在32摄氏度时具有31千卡/摩尔三聚体（130千焦/摩尔三聚体）的最大稳定性（内在自由能）。在该温度范围内，去折叠时的热容变化ΔCp和m值被认为是恒定的，分别计算为2.86千卡/摩尔三聚体（11.9千焦/摩尔三聚体）和5.67千卡/摩尔三聚体·M（23.7千焦/摩尔三聚体·M）。研究了三聚化对热力学稳定性的影响。定量描述了热稳定性的几个相互关联的方面，如去折叠动力学、自由能的温度依赖性以及变性蛋白分数的浓度和温度依赖性。特别讨论了嗜酸热硫化叶菌腺苷酸激酶以及一般寡聚蛋白的吉布斯 - 亥姆霍兹函数的性质，并与单体蛋白类似函数的性质进行了比较。此外，我们还讨论了方法学方面：我们得到了同三聚体蛋白变性剂去折叠等温线的解析表达式；我们在附录中包含了所用表达式推导的公式。
