A native-like three-α-helix bundle protein from structure-based redesign:: A novel maquette scaffold
A native-like three-α-helix bundle protein from structure-based redesign:: A novel maquette scaffold
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DOI：
10.1021/ja973538m
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发表时间：
1998-04-29
影响因子：
15
通讯作者：
Dutton, PL
中科院分区：
文献类型：
--
作者：
Johansson, JS;Gibney, BR;Dutton, PL
以晶体学特征的三链卷曲螺旋“Coil-Ser”为起点，设计并化学合成了结构独特的65个氨基酸螺旋-环-螺旋'-环-螺旋“三α螺旋束α(3)-1。α(3)-1的圆二色光谱具有典型的α螺旋特征， a θ(222) = -22 600 deg.cm(2).dmol(-1)，表明α螺旋含量为80.5％。沉降平衡分析超速离心表明α(3)-1 在溶液中是单体。与设计参数一致，独特的疏水核心色氨酸残基的荧光发射最大值出现在 324 纳米。通过 CD 光谱测量，基于可逆盐酸胍变性评估的 Delta G(H2O) 为 -4.6 +/- 0.3 kcal.mol(-1) (m = 2.2 +/- 0.2 kcal.mol-(1).M-1)。 α(3)-1 的 H-1-NMR 谱的酰胺-芳香族区域显示出优异的化学位移 色散和分辨率。所有 35 个预期的甲基相关性均在 C-13-HSQC 谱中得到解释，为类天然疏水核心的存在提供了严格的证据。 α(3)-1 的单体性质应有助于当前设计以及具有工程化结合位点的未来变体的 NMR 结构研究和动力学蛋白质折叠分析。这个单链的实用性 正在探索用于扩大模型中结合的生化辅因子范围的三a螺旋束框架。
A uniquely structured 65 amino acid helix-loop-helix'-loop-helix " three-alpha-helix bundle, alpha(3)-1, was designed and chemically synthesized, using the crystallographically characterized three stranded coiled coil "Coil-Ser", as a starting point. The circular dichroism spectrum of alpha(3)-1 has a typical alpha-helical signature, with a [theta](222) = -22 600 deg.cm(2).dmol(-1), indicating a 80.5% alpha-helical content. Sedimentation equilibrium analytical ultracentrifugation revealed that alpha(3)-1 is monomeric in solution. Consistent with the design parameters, the fluorescence emission maximum of the unique hydrophobic core tryptophan residue occurs at 324 nm. The evaluated Delta G(H2O) based on reversible guanidine hydrochloride denaturation is -4.6 +/- 0.3 kcal.mol(-1) (m = 2.2 +/- 0.2 kcal.mol-(1).M-1) as measured by CD spectroscopy. The amide-aromatic region of the H-1-NMR spectrum of alpha(3)-1 illustrates excellent chemical shift dispersion and resolution. All 35 expected methyl correlations are accounted for in the C-13-HSQC spectrum, providing stringent evidence for the existence of a native-like hydrophobic core. The monomeric nature of alpha(3)-1 should facilitate NMR structural studies and kinetic protein folding analysis of the current design, and on future variants with engineered binding sites. The utility of this single-chain three-a-helix bundle framework for expanding the range of biochemical cofactors bound in maquettes is being explored.