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首页> 外文期刊>Journal of Molecular Biology >Thermodynamics and kinetics of unfolding of the thermostable trimeric adenylate kinase from the archaeon Sulfolobus acidocaldarius
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Thermodynamics and kinetics of unfolding of the thermostable trimeric adenylate kinase from the archaeon Sulfolobus acidocaldarius

机译：来自古细菌Sulfolobus acidocaldarius的热稳定三聚腺苷酸激酶解折叠的热力学和动力学

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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 Delta C-p and the In-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. (C) 1998 Academic Press. [References: 65]
机译：利用变性剂诱导的展开，差示扫描量热法，圆二色性光谱和酶灭活研究，全面表征了嗜热古细菌Sulfolobus acidocaldarius的腺苷酸激酶的热稳定性。由于聚集，蛋白质的热诱导解折叠是不可逆的，而氯化胍诱导的解折叠是可逆的。已知该蛋白质在其天然状态下是同源三聚体，并且我们确定了在变性剂解折叠的情况下，它在解离后会解折叠。我们使用变性剂展开法在5至70摄氏度的温度范围内测量了蛋白质的热力学稳定性。该蛋白质在32摄氏度（基于线性外推模型）下的最大稳定性（固有自由能）为31 kcal / mol-三聚体（130 kJ / mol-三聚体）。在此温度范围内，展开ΔCp时的热容变化和In值被认为是恒定的，计算得出为2.86 kcal / mol-三聚体（11.9 kJ / mol-三聚体）和5.67 kcal / mol-三聚体M（23.7 kJ / mol-三聚体M）。研究了三聚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，例如展开动力学，自由能的温度依赖性以及变性蛋白质部分的浓度和温度依赖性。一般而言，讨论了嗜酸链球菌的腺苷酸激酶和寡聚蛋白的吉布斯-亥姆霍兹功能的性质，并与单体蛋白的类似功能的性质进行了比较。此外，我们讨论了方法方面的问题：我们获得了同源三聚体蛋白变性展开等温线的分析表达;我们提供了一个公式附录，其中包含所用表达式的派生词。 （C）1998年学术出版社。 [参考：65]

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