Crystal polymorphism serves as a strategy to study the conformational flexibility of proteins. However, the relationship between protein crystal packing and protein conformation often remains elusive. In this study, two distinct crystal forms of a green fluorescent protein variant, NowGFP, are compared: a previously identified monoclinic form (space group C2) and a newly discovered orthorhombic form (space group P212121). Comparative analysis reveals that both crystal forms exhibit nearly identical linear assemblies of NowGFP molecules interconnected through similar crystal contacts. However, a notable difference lies in the stacking of these assemblies: parallel in the monoclinic form and perpendicular in the orthorhombic form. This distinct mode of stacking leads to different crystal contacts and induces structural alteration in one of the two molecules within the asymmetric unit of the orthorhombic crystal form. This new conformational state captured by orthorhombic crystal packing exhibits two unique features: a conformational shift of the β-barrel scaffold and a restriction of pH-dependent shifts of the key residue Lys61, which is crucial for the pH-dependent spectral shift of this protein. These findings demonstrate a clear connection between crystal packing and alternative conformational states of proteins, providing insights into how structural variations influence the function of fluorescent proteins.

中文翻译：

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NowGFP 的两种晶体多晶型物的比较揭示了一种被晶体堆积捕获的新构象状态。


晶体多态性是研究蛋白质构象灵活性的一种策略。然而，蛋白质晶体堆积和蛋白质构象之间的关系通常仍然难以捉摸。在这项研究中，比较了绿色荧光蛋白变体 NowGFP 的两种不同晶体形式：先前确定的单斜晶系形式（空间群 C2）和新发现的斜方晶系形式（空间群 P212121）。比较分析表明，两种晶型表现出几乎相同的 NowGFP 分子线性组装体，这些分子通过相似的晶体接触互连。然而，一个显着的区别在于这些组件的堆叠：单斜晶系形式是平行的，正交系形式是垂直的。这种独特的堆叠模式导致不同的晶体接触，并诱导斜方晶型的不对称单元内两个分子之一的结构改变。斜方晶体填充捕获的这种新构象状态表现出两个独特的特征：β桶支架的构象变化和关键残基 Lys61 的 pH 依赖性变化的限制，这对于该蛋白质的 pH 依赖性光谱变化至关重要。这些发现证明了晶体堆积与蛋白质的替代构象状态之间的明确联系，为结构变化如何影响荧光蛋白的功能提供了见解。