STRUCTURAL AND THERMODYNAMIC CONSEQUENCES OF BURYING A CHARGED RESIDUE WITHIN THE HYDROPHOBIC CORE OF T4 LYSOZYME
STRUCTURAL AND THERMODYNAMIC CONSEQUENCES OF BURYING A CHARGED RESIDUE WITHIN THE HYDROPHOBIC CORE OF T4 LYSOZYME
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DOI：
10.1021/bi00113a006
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发表时间：
1991-12-10
期刊：
影响因子：
2.9
通讯作者：
MATTHEWS, BW
中科院分区：
文献类型：
--
作者：
DAOPIN, S;ANDERSON, DE;MATTHEWS, BW
为了确定在蛋白质核心放置带电基团的能量和结构后果，在噬菌体T4溶菌酶中构建了两个“埋藏电荷”突变体，Met 102 -> Lys （M102K）和Leu 133 -> Asp （LI33D）。这两种蛋白质在中性pH下折叠，尽管它们比野生型稳定得多。M102K的活性约为野生型的35%，而L133D的活性约为4%。M102K可以结晶，并在高分辨率下确定了其结构。该突变体的晶体结构（pH值为6.8）与野生型非常相似，除了α -螺旋包含残基108-113之外。在野生型溶菌酶中，这个螺旋的一侧暴露在溶剂中，另一侧接触Met 102。在M102K结构中，这个α -螺旋变得更加可移动，可能允许Lys 102部分进入溶剂。M102K的稳定性，通过监测蛋白质与CD的展开来确定，是ph依赖性的，与取代的氨基酸的带电形式比不带电形式更不稳定一致。通过差异滴定和核磁共振分析，估计赖氨酸102的pK(a)为6.5，所有赖氨酸的C（epsilon）位置都含有C-13。当pH值低于6.5时，M102K在室温下的整体三维结构似乎保持在pH值为3左右，尽管有证据表明一些结构调整可能允许溶剂接近赖氨酸102的质子化形式。
To determine the energetic and structural consequences of placing a charged group within the core of a protein, two "buried charge" mutants, Met 102 --> Lys (M102K) and Leu 133 --> Asp (LI33D) were constructed in phage T4 lysozyme. Both proteins fold at neutral pH, although they are substantially less stable than wild type. The activity of M102K is about 35% that of wild type, while that of L133D is about 4%. M102K could be crystallized, and its structure was determined at high resolution. The crystal structure (at pH 6.8) of the mutant is very similar to that of wild type except for the alpha-helix that includes residues 108-113. In wild-type lysozyme, one side of this helix is exposed to solvent and the other contacts Met 102. In the M102K structure this alpha-helix becomes much more mobile, possibly allowing partial access of Lys 102 to solvent. The stability of M102K, determined by monitoring the unfolding of the protein with CD, is pH-dependent, consistent with the charged form of the substituted amino acid being more destabilizing than the uncharged form. The pK(a) of Lys 102 was estimated to be 6.5 both by differential titration and also by NMR analysis of isotopically labeled protein with C-13 incorporated at the C(epsilon) position of all lysines. As the pH is lowered below pH 6.5, the overall three-dimensional structure of M102K at room temperature appears to be maintained to pH 3 or so, although there is evidence for some structural adjustment possibly allowing solvent accessibility to the protonated form of Lys 102.