Kumar Rajesh, Bhuyan Abani K
School of Chemistry, University of Hyderabad, Hyderabad, 500046, India.
J Biol Inorg Chem. 2009 Jan;14(1):11-21. doi: 10.1007/s00775-008-0420-5. Epub 2008 Aug 28.
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.
为了探究蛋白质与盐酸胍（GdnHCl）结合相互作用后其内部动力学和功能的变化，采用激光闪光光解和光吸收方法，在pH 7、22℃条件下，研究了存在亚变性浓度GdnHCl时马肌红蛋白-CO复合物（MbCO）中的动态事件。通过对MbCO中的CO进行激光光解测量了双分子复合和解离的速率系数（k(on)），并使用六氰合铁离子或NO通过CO置换法测定了CO解离速率（k(off)）。从天然状态开始，在存在0.1 - 0.3 M GdnHCl时，k(on)和k(off)的值下降约1.4（±0.1）倍，然后在更高浓度的变性剂下增加。这被视为蛋白质在亚变性区域内存在内部运动限制和稳定性增加的证据，导致光解的CO从溶剂中有门控进入。双分子复合的速率在速率与GdnHCl浓度的图中未显示出任何下降。作为GdnHCl浓度函数测量的CO解离反应的活化焓值和相对于天然状态熵的熵损失值表明，蛋白质在亚变性条件下确实得到了稳定。对存在不同浓度GdnHCl时肌红蛋白的热解折叠转变分析表明，从线性自由能模型得出的该蛋白质稳定性比真实稳定性约低3 - 4 kcal mol(-1)。结果表明变性剂结合模型适用于分析GdnHCl诱导的解折叠数据，并给出了该蛋白质真实稳定性为7.9 kcal mol(-1)的值。
