Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride
Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride
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DOI：
10.1007/s00775-008-0420-5
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发表时间：
2009-01-01
影响因子：
3
通讯作者：
Bhuyan, Abani K.
中科院分区：
文献类型：
--
作者：
Kumar, Rajesh;Bhuyan, Abani K.
为了了解蛋白质与盐酸胍（GdnHCl）结合后的内部动力学和功能变化，采用激光闪光光解和光吸收方法研究了马肌红蛋白-一氧化碳复合物（MbCO）在亚变性浓度的盐酸胍（GdnHCl）存在下的动力学事件。通过激光光解MbCO中的CO来测量双生子再结合和双分子再结合的速率系数（k（on）），并且通过使用六氰基铁离子或NO的CO置换法来确定CO解离速率（k（off））。在0.1-0.3 M GdnHCl存在下，k（on）和k（off）的值降低约1.4（+/-0.1）倍，然后在更高浓度的变性剂下增加。这已被视为证据的内部运动的限制和增加的稳定性的蛋白质在subdenaturing区域引起门控进入的光解CO从溶剂中。成对再结合的速率在速率对GdnHCl浓度图中未显示任何降低。CO解离反应的活化焓和相对于天然状态熵的熵损失的值，都作为GdnHCl浓度的函数测量，表明蛋白质确实是稳定的subdenaturing条件下。对肌红蛋白在不同浓度盐酸钆存在下的热去折叠转变的分析表明，从线性自由能模型中提取的这种蛋白质的稳定性比真实稳定性低约3-4 kcal mol（-1）。结果表明，适当的变性剂结合模型的GdnHCl诱导的解折叠数据的分析，并提供了一个值为7.9 kcal mol（-1）的蛋白质的真实稳定性。
To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin-CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 degrees C. The rate coefficients for geminate rebinding and bimolecular rebinding (k (on)) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k (off)) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k (on) and k (off) decrease by approximately 1.4 (+/- 0.1)-fold in the presence of 0.1-0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3-4 kcal mol(-1) less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol(-1) as the true stability of the protein.