Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.
Analysis of the two-state behavior of the thermal unfolding serum retinol binding protein containing a single retinol ligand.
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分析含有单一视黄醇配体的热解折叠血清视黄醇结合蛋白的两种状态行为。
DOI：
10.1021/bi00139a019
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Brouillette,CG
中科院分区：
文献类型：
--
作者：
Muccio,DD;Waterhous,DV;Fish,F;Brouillette,CG
化学系，亚拉巴马大学伯明翰分校，伯明翰，亚拉巴马35294，和有机化学和生物化学研究系，南方研究所，伯明翰，亚拉巴马35205，1991年9月3日接收;修订后的手稿，1992年3月19日接收摘要：通过使用CD和DSC，在pH 7.4下研究了含有单个紧密结合的视黄醇配体的全血清视黄醇结合蛋白的热去折叠。全蛋白（[视黄醇]/[蛋白质]= 1）的DSC吸热在78 ℃的转变温度附近是不对称的。利用230 nm处椭圆率的变化，热去折叠曲线也关于以78 ℃附近为中心的拐点不对称。通过三种方法测定范氏霍夫焓，并与200千卡/摩尔的量热焓（Ai/cai）进行比较。从转变温度对配体结合蛋白质浓度的依赖性确定范特霍夫焓为190千卡/摩尔。该值与从DSC曲线（f = 184 kcal/mol）和光谱曲线（A//vH= 181 kcal/mol）拟合到包括配体解离的两态热力学模型（NR^ U+ R，其中NR是天然全蛋白，U是未折叠的脱辅基蛋白，R是视黄醇）中发现的范特霍夫谱一致。用忽略配体解离（N^ U）的两态模型获得的一致性较差。此外，NR^ U+ R模型解释了CD和DSC转变中的不对称性，并产生了比NU模型更好的数据拟合。从这些考虑和对其他平衡模型的模拟中，我们认为NR^ U+ R模型是描述这种配体结合蛋白质热去折叠的最简单模型。由DSC和CD数据拟合得到的平均van 't霍夫焓表明，该过程的协同系数为0.925，表明该全蛋白的去折叠近似为两态。
Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, and Department of Organic Chemistry and Biochemistry Research, Southern Research Institute, Birmingham, Alabama 35205 Received September 3, 1991; Revised Manuscript Received March 19, 1992 abstract: Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein]= 1) was asymmetric about the transition temperature of 78 C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 C. van’t Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (Ai/cai) of 200 kcal/mol. A van’t Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van’t Hoff enthalpies found from fits of the DSC (/= 184 kcal/mol) and spectroscopic (A//vH= 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR^ U+ R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N^ U). Furthermore, the NR^ U+ R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR^ U+ R model is the simplest model that describes thethermal unfolding of this ligand-bound protein. Using an averaged van’t Hoff enthalpy determined from fits of DSC and CD data, the cooperativity of this process was 0.925, indicating that the unfolding of the holoprotein is nearly two-state.
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