Kono H, Saito M, Sarai A
Tsukuba Life Science Center, The Institute of Physical & Chemical Research (RIKEN), Ibaraki, Japan.
Proteins. 2000 Feb 1;38(2):197-209. doi: 10.1002/(sici)1097-0134(20000201)38:2<197::aid-prot8>3.0.co;2-b.
We have analyzed the effect of cavity-filling mutations on protein stability by means of free-energy calculations based on molecular dynamics simulations to identify the factors contributing to stability changes caused by the mutations. We have studied the DNA-binding domain of Myb, which has a cavity in one of three homologous repeat units, and analyzed a series of mutations with nonnatural and natural amino acids at a single site, which change the size of the cavity. We found that the calculated free-energy changes caused by the mutations are in excellent agreement with experimental data (correlation coefficient 0.98). The free-energy changes in the native and denatured states were independently compared with the unfolding free-energy change (deltadeltaG) and cavity-volume changes (deltaV), and it was found that deltadeltaG and deltaV correlate with the native-state free-energy changes but not with the denatured-state free-energy changes. Further analyses in terms of enthalpy and entropy show that compensation between entropy and enthalpy occurs in the denatured state but not in the native state. The main contribution to the native-state free energy was found to be van der Waals interactions associated with the cavity. We estimate that the decrease in free energy per methylene group, which results from filling the cavity, is about 2 to 3 kcal/mol. These results suggest that the stabilization of a protein by cavity-filling mutations be determined primarily by the free energy associated with the cavity volume in the native state.
我们通过基于分子动力学模拟的自由能计算，分析了填充空腔突变对蛋白质稳定性的影响，以确定导致突变引起稳定性变化的因素。我们研究了Myb的DNA结合结构域，该结构域在三个同源重复单元之一中存在一个空腔，并分析了在单个位点上一系列用非天然和天然氨基酸进行的突变，这些突变改变了空腔的大小。我们发现，由突变引起的计算出的自由能变化与实验数据高度吻合（相关系数为0.98）。将天然态和变性态的自由能变化分别与解折叠自由能变化（deltadeltaG）和空腔体积变化（deltaV）进行比较，发现deltadeltaG和deltaV与天然态自由能变化相关，而与变性态自由能变化无关。进一步从焓和熵的角度分析表明，熵和焓之间的补偿发生在变性态而非天然态。发现对天然态自由能的主要贡献是与空腔相关的范德华相互作用。我们估计，由于填充空腔导致的每个亚甲基自由能的降低约为2至3千卡/摩尔。这些结果表明，通过填充空腔突变实现的蛋白质稳定主要由天然态中空腔体积相关的自由能决定。
