Burnett J C, Botti P, Abraham D J, Kellogg G E
Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298-0133, USA.
Proteins. 2001 Feb 15;42(3):355-77. doi: 10.1002/1097-0134(20010215)42:3<355::aid-prot60>3.0.co;2-f.
A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.
报道了一种用于与蛋白质突变相关的自由能变化分子建模的实用计算方法。详细描述了多种脱氧和氧合血红蛋白突变体的从头生成、优化及热力学分析。血红蛋白被证明是进行研究的理想候选蛋白质，因为其天然脱氧和氧合状态均已通过晶体学确定，并且其大量且多样的突变体群体已进行了热力学表征。使用分子建模程序HINT（亲水性相互作用）对所有通过计算生成的血红蛋白突变体的非共价相互作用进行定量研究。HINT对所有生物分子非共价相互作用进行评分，包括氢键、酸碱、疏水-疏水、酸-酸、碱-碱以及疏水-极性相互作用，以生成二聚体-二聚体界面“分数”，这些分数被转化为自由能估计值。对23种脱氧和氧合状态的血红蛋白突变体的分析表明，突变残基对结构结合水的影响（反之亦然）对于生成准确的自由能估计值很重要。对于几种突变体，结构水的添加/消除是理解残基突变热力学后果的关键。脱氧血红蛋白突变体的计算数据与实验数据之间具有良好的一致性（r = 0.79，斜率 = 0.78，标准误差 = 1.4 kcal mol⁻¹，n = 23）。最初对氧合血红蛋白突变体的估计不太准确（r = 0.48，斜率 = 0.47，标准误差 = 1.4 kcal mol⁻¹，n = 23）。然而，从该数据集中剔除三个异常值后，相关性得到改善，r = 0.87（斜率 = 0.72，标准误差 = 0.75，n = 20）。这三个突变可能会使血红蛋白四级结构受到显著扰动，超出了我们结构优化程序的范围。所描述的方法在检查蛋白质结构中的残基电离状态时也很有用。具体而言，我们发现天然脱氧血红蛋白二聚体-二聚体界面内的一个酸性残基在生理pH下可能被质子化。最后的分析是对新型血红蛋白突变体的模型设计，这些突变体可改变协同自由能（ΔGc）——从脱氧血红蛋白到氧合血红蛋白变构转变的能量屏障。
