EVALUATION OF CONTRIBUTIONS FROM ELECTROSTATIC INTERACTIONS TO THERMODYNAMIC QUANTITIES OF PROTEINS

为了评估蛋白质分子表面离子电荷之间的库仑相互作用对其结构稳定性的贡献，我们在去年的工作基础上进行了计算和实验研究：(1)计算研究：对于分子表面离子电荷部分中和的静电突变体，利用微分泊松-玻尔兹曼方程估计其库仑相互作用能U_<c->，将这种“部分中和分析”方法应用于细胞色素c和泛素，得到以下结果：(1)在中性pH和低离子强度下，部分中和的金枪鱼细胞色素c分子种上的U_c的平均值U初始减小；在n=6时达到最小值，净电荷为+3，然后随着中和数n的增加而增加。这一预测与细胞色素c分子的构象随着g . n. c的增加而从展开形态变为MG形态的观察结果吻合得很好。特别是Lysl3-Glu9O和lys53 - asp50o这两个离子对，由于盐离子的屏蔽作用，占据了库仑能的主导地位。(3)中和泛素的酸性残基和碱性残基对U_c的分布有相似的影响。(2)实验研究：首先，我们制备了部分中和的细胞色素c样品，并对其进行了相对于n的纯化。我们使用高精度差示扫描量热计（DSC）测量了n = 3-5样品的热容函数C_p(T)。(1)修饰蛋白的热跃迁引起的C_p(T)的吸热峰比天然细胞色素c的吸热峰宽，这是由于在中和的分子集合中，存在不同位置中和的不同分子种，具有不同的静电能。(2) C_p(T)值在热转变温度以上减小，表明分子内发生了一些内聚相互作用，几乎不受蛋白质浓度的影响。少

To evaluate contributions from Coulomb interactions between ionic charges on the surface of protein molecules to their structural stability, we have made both computational and experimental studies following the work of last year :(1)Computational studies : For electrostatic mutants whose ionic charges on the molecular surface are partially neutralized, differential Poisson-Boltzmann equations were solved to estimate their Coulomb-interaction energies U_<c-> Applying this 'Partial Neutralization Analysis' method to cytochrome c and ubiquitin, we obtained following results :(1)At neutral pH and low ionic strength, the average value U of U_c taken over the molecular species of partially neutralized tuna cytochrome c decreases initially, reaches a minimum at n=6 with+3 net charges, and then increases with increasing neutralization number n. This prediction agrees well with the observation that cytochrome c molecules change their conformation from the unfolded to the MG form with increasin … More g n. c At high ionic strength, the distribution of U_c has three distinct peaks, and U increases monotonously with n, This is caused by the fact that the contribution from interactions between nearby ionic residues, especially those of the two ion pairs, Lysl3-Glu9O and Lys53-Asp5O, dominate the Coulomb energy due to the shielding effect of salt ions. (3)Neutralizing acidic and basic residues of ubiquitin was found to have similar effects on the distribution of U_c.(2)Experimental studies : First, we prepared partially neutralized samples of cytochrome c and purified them with respect to n. Using a high precision differential scanning calorimeter(DSC), we have measured heat capacity functions C_p(T)for the samples with n = 3-5. (1)The heat-absorption peak of C_p(T)due to thermal transition of modified proteins is broader than that of natural cytochrome c. This wil1 originate from the fact that, in the ensemble of neutralized molecules, there exist various molecular species that are neutralized at different sites and have different electrostatic energies. (2)The value of C_p(T)decreases above the temperature of thermal transition, which suggests that some intramolecular cohesive interactions are made as it is nearly independent of protein concentration. Less