CONTRIBUTIONS OF THE LARGE HYDROPHOBIC AMINO-ACIDS TO THE STABILITY OF STAPHYLOCOCCAL NUCLEASE
CONTRIBUTIONS OF THE LARGE HYDROPHOBIC AMINO-ACIDS TO THE STABILITY OF STAPHYLOCOCCAL NUCLEASE
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DOI：
10.1021/bi00487a007
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发表时间：
1990-09-04
期刊：
影响因子：
2.9
通讯作者：
MEEKER, AK
中科院分区：
文献类型：
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作者：
SHORTLE, D;STITES, WE;MEEKER, AK
为了定量金黄色葡萄球菌核酸酶中大的疏水残基对其天然状态稳定性的贡献，对11个亮氨酸、9个缬氨酸、7个酪氨酸、5个异亮氨酸、4个蛋氨酸和3个苯丙氨酸残基进行了定点突变，构建了单一的丙氨酸和甘氨酸取代基。此外，每一种异亮氨酸也被突变为缬氨酸。最终收集的83个突变核酸酶被提交给使用本征色氨酸荧光的盐酸胍变性，以监测天然状态和变性状态之间的平衡常数。通过对这些数据的分析，得到了S突变蛋白对可逆变性的稳定性(ΔGH2O)和对盐酸胍的敏感性(mGaHCl或d(ΔG)/d[GuHCl])。观察到了四个意想不到的趋势。(1)发现改变mGuHCl的突变位点具有显著的两部分分布：增加这一参数的突变只涉及为主要疏水核心贡献侧链的残基，这些残基以五链β-桶为中心，而导致mGuHCl减少的突变聚集在第二个更小且定义不太明确的疏水核心周围。(2)根据疏水侧链从水到正辛醇的转移自由能估算，六种残基中每一类突变体的平均稳定性损失比估算的要大2-3倍。(3)丙氨酸或甘氨酸取代一种特定氨基酸时，其稳定性损失的大小在氨基酸的不同出现部位之间差别很大，这表明特定残基周围的环境决定了它的侧链对稳定性的贡献有多大。在统计分析的基础上，提供对这种环境影响的最佳估计的参数为Cα的数目。半径为10的球体内的碳。(4)mGuHCl变化的绝对值与失稳显著相关。这种相关性有力地支持了这样的结论，即氨基酸取代可以通过影响蛋白质变性状态的结构和自由能来间接破坏蛋白质的稳定性。
To quantitate the contributions of the large hydrophobic residues in staphylococcal nuclease to the stability of its native state, single alanine and glycine substitutions were constructed by site-directed mutagenesis for each of the 11 leucine, 9 valine, 7 tyrosine, 5 isoleucine, 4 methionine, and 3 phenylalanine residues. In addition, each isoleucine was also mutated to valine. The resulting collection of 83 mutant nucleases was submitted to guanidine hydrochloride denaturation using intrinsic tryptophan fluorescence to monitor the equilibrium constant between the native and denatured states. From analysis of these data, each mutant protein''s stability to reversible denaturation (.DELTA.GH2O) and sensitivitiy to guanidine hydrochloride (mGaHCl or d(.DELTA.G)/d[GuHCl]) were obtained. Four eunexpected trends were observed. (1) A striking bipartite distribution was found for sites of mutations that altered mGuHCl: mutations that increased this parameter only involved residues that contribute side chains to the major hydrophobic core centered around a five-strand .beta.-barrel, whereas mutations that caused mGuHCl to decrease clustered around a second, smaller and less well-defined hydrophobic core. (2) The average stability loss for mutants in each of the six residue classes was 2-3 times greater than that estimated on the basis of the free energy of transfer of the hydrophobic side chain from water to n-octanol. (3) The magnitude of the stability loss on substituting Ala or Gly for a particular type of amino acid varied extensively among the different sites of its occurrence in nulease, indicating that the environment surrounding a specific residue determines how large a stability contribution its side chain will make. On the basis of statistical analyses, the parameter that provided the best estimate of this environmental effect on .DELTA..DELTA.G is the number of C.alpha. carbons within a sphere of 10-.ANG. radius. (4) A significant correlation was found between the absolute value of the change in mGuHCl and the loss of stability. This correlation strongly supports the conclusion that amino acid substitutions can destabilize a protein indirectly via their effects on the structure and free energy of the denatured state.