The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability.
The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability.
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DOI：
10.1093/protein/7.11.1365
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发表时间：
1994-11
期刊：
影响因子：
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通讯作者：
N. Zhou;C. Kay;R. Hodges
中科院分区：
文献类型：
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作者：
N. Zhou;C. Kay;R. Hodges
螺旋间静电吸引对线圈稳定性的净能量贡献已经用从头设计的合成线圈进行了量化。合成的模型卷曲线圈（EK），用e和g位置的氨基酸残基表示，只包含螺旋间离子相互作用，不包含任何可能的（i, i + 3）和（i, i + 4）螺旋内离子相互作用，由两个相同的35个残基多肽链组成，具有七重位重复KgLaG-bAcLdEeKf。制备了3个突变体，将EK中e个位置的5个Glu残基突变为Gln残基（QK）；g位的5个Lys残基被改变为Gln残基（EQ）或这些突变在e和g位都发生（QQ）。通过测量在220 nm处的椭圆度作为20℃下尿素浓度的函数来确定四种螺旋线圈的稳定性。通过双突变循环分析，可以将螺旋间离子吸引力对螺旋线圈稳定性的能量贡献与其他贡献（如螺旋偏好和疏水性）分离出来。通过三个独立的比较，得出一个螺旋间离子对线圈稳定性的能量贡献为0.37 +/- 0.01 kcal/mol。这一发现表明，蛋白质表面大量的弱螺旋间静电相互作用可以对蛋白质的稳定性做出实质性的贡献。此外，还测定了单个突变E（-）—>Q、K（+）—>Q、Q—>E度和E（-）—>E度的能量贡献（δ δ G分别为0.22、0.26、0.46和0.65 kcal/mol）。（摘要删节250字）
The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability has been quantitated using de novo designed synthetic coiled-coils. The synthesized model coiled-coil (EK), denoted by amino acid residues in positions e and g, which contains only interhelical ionic interactions without any possible (i, i + 3) and (i, i + 4) intrahelical ionic interaction, consists of two identical 35 residue polypeptide chains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coils were prepared where five Glu residues at e positions in EK were mutated to Gln residues (QK); five Lys residues at g positions were altered to Gln residues (EQ) or these mutations were effected at both positions e and g (QQ). The stabilities of the four coiled-coils were determined by measuring the ellipticities at 220 nm as a function of urea concentration at 20 degrees C. By using a double-mutant cycle analysis it was possible to isolate the energetic contribution of interhelical ionic attractions to coiled-coil stability from the other contributions such as helical preference and hydrophobicity. The 0.37 +/- 0.01 kcal/mol of energetic contribution of one interhelical ion pair to the coiled-coil stability was obtained from three independent comparisons. This findings suggests that a large number of weak interhelical electrostatic interactions on the surface of a protein can make a substantial contribution to protein stability. In addition, the energetic contributions of a single mutation E(-)-->Q, K(+)-->Q, Q-->E degrees and E(-)-->E degrees were also determined (delta delta G = 0.22, 0.26, and 0.46 and 0.65 kcal/mol for the single mutations, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)