Attempts to create novel ligand-binding proteins often focus on formation of a binding pocket with shape complementarity against the desired ligand (particularly for compounds that lack distinct polar moieties). Although designed proteins often exhibit binding of the desired ligand, in some cases they display unintended recognition behavior. One such designed protein, that was originally intended to bind tetrahydrocannabinol (THC), was found instead to display binding of 25-hydroxy-cholecalciferol (25-D3) and was subjected to biochemical characterization, further selections for enhanced 25-D3 binding affinity and crystallographic analyses. The deviation in specificity is due in part to unexpected altertion of its conformation, corresponding to a significant change of the orientation of an α-helix and an equally large movement of a loop, both of which flank the designed ligand-binding pocket. Those changes led to engineered protein constructs that exhibit significantly more contacts and complementarity towards the 25-D3 ligand than the initial designed protein had been predicted to form towards its intended THC ligand. Molecular dynamics simulations imply that the initial computationally designed mutations may contribute to the movement of the helix. These analyses collectively indicate that accurate prediction and control of backbone dynamics conformation, through a combination of improved conformational sampling and/or de novo structure design, represents a key area of further development for the design and optimization of engineered ligand-binding proteins.

中文翻译：

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蛋白质折叠的不可预测的可塑性促进了工程化的配体结合蛋白的意想不到的特异性。

尝试创建新的配体结合蛋白通常集中在形成与所需配体形状互补的结合袋中（特别是对于缺少独特极性部分的化合物）。尽管设计的蛋白质通常表现出所需配体的结合，但在某些情况下，它们表现出意外的识别行为。发现这样一种设计的蛋白质原本旨在与四氢大麻酚（THC）结合，但与25-羟基胆钙化醇（25-D3）结合，并进行了生化表征，进一步选择了增强的25-D3结合亲和力和晶体学分析。特异性的差异部分是由于其构象的意外变化所致，这对应于α螺旋方向的显着变化和环的同等大的运动，两者均位于设计的配体结合口袋的侧面。这些变化导致工程蛋白构建体对25-D3配体的接触和互补性比预期的初始设计蛋白对预期的THC配体的显着提高。分子动力学模拟表明，最初的计算设计突变可能有助于螺旋的运动。这些分析共同表明，通过改进的构象采样和/或从头结构设计的组合，对骨架动力学构象的准确预测和控制代表了工程设计的配体结合蛋白设计和优化的进一步发展的关键领域。这些变化导致工程蛋白构建体对25-D3配体的接触和互补性显着高于最初设计的蛋白质对其预期的THC配体的形成。分子动力学模拟表明，最初的计算设计突变可能有助于螺旋的运动。这些分析共同表明，通过改进的构象采样和/或从头进行结构设计的组合，对骨架动力学构象的准确预测和控制代表了工程设计的配体结合蛋白的设计和优化的进一步发展的关键领域。这些变化导致工程蛋白构建体对25-D3配体的接触和互补性显着高于最初设计的蛋白质对其预期的THC配体的形成。分子动力学模拟表明，最初的计算设计突变可能有助于螺旋的移动。这些分析共同表明，通过改进的构象采样和/或从头进行结构设计的组合，对骨架动力学构象的准确预测和控制代表了工程设计的配体结合蛋白的设计和优化的进一步发展的关键领域。分子动力学模拟表明，最初的计算设计突变可能有助于螺旋的运动。这些分析共同表明，通过改进的构象采样和/或从头进行结构设计的组合，对骨架动力学构象的准确预测和控制代表了工程设计的配体结合蛋白的设计和优化的进一步发展的关键领域。分子动力学模拟表明，最初的计算设计突变可能有助于螺旋的运动。这些分析共同表明，通过改进的构象采样和/或从头进行结构设计的组合，对骨架动力学构象的准确预测和控制代表了工程设计的配体结合蛋白的设计和优化的进一步发展的关键领域。