Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold.
Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold.
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DOI：
10.1093/protein/gzm070
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发表时间：
2008-02
期刊：
影响因子：
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通讯作者：
Maya Bar;T. Scherf;D. Fass
中科院分区：
文献类型：
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作者：
Maya Bar;T. Scherf;D. Fass
我们测试了富含二硫键的抗冻蛋白作为潜在的支架，用于设计或选择具有结合周期性组织表面的能力的蛋白质。天然抗冻蛋白是一种β-螺旋，其冰结合面上具有极其规则的苏氨酸侧链二维网格。在这个面上进行氨基酸替换，用跨越β-折叠倾向范围的其他氨基酸替换天然苏氨酸块。通过质谱、反相高效液相色谱、硫醇反应性、圆二色性和核磁共振光谱对显示不同官能团阵列的变体进行研究，以评估其相对于野生型的结构和稳定性。尽管这些具有 16 个半胱氨酸的 84 个残基蛋白质固有地存在错误折叠的可能性，但这些突变体在细菌中得到了良好的表达。我们证明大多数突变体基本上保留了天然折叠。这种二硫键键合的β-螺旋支架具有热稳定性和对氨基酸取代的显着耐受性，因此可用于设计和工程具有结合各种靶向有序材料表面潜力的大分子。
We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.