Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions
Nonclassical Pathways of Protein Crystallization in the Presence of Multivalent Metal Ions
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DOI：
10.1021/cg501099d
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发表时间：
2014-12-01
影响因子：
3.8
通讯作者：
Schreiber, Frank
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文献类型：
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作者：
Sauter, Andrea;Oelker, Melanie;Schreiber, Frank
以牛β-乳球蛋白为模型体系，研究了二价和三价盐ZnCl2和YCl3存在下的结晶过程。先前的工作表明，在YCl3存在的情况下，蛋白质溶液经历了重入凝聚(RC)相行为，即在两个边界盐浓度c*<；c**之间发生凝聚相。在氯化锌存在的情况下，c*也存在，但高盐浓度的蛋白质溶液在较高的蛋白质浓度(>；20 mg/mL)下并不完全透明。因此，我们将这种扩散转变称为伪c**。小角X射线散射测量表明，在c*附近，有效相互作用由排斥变为吸引，而在赝色c**附近，吸引作用占主导地位。C*附近和伪c**附近的溶液为生长高质量的蛋白质单晶提供了优化的条件，但途径不同。C*附近的晶体生长遵循经典的一步成核过程，而准c**(对于锌)或c**(对于Y)的结晶遵循非经典过程，在结晶之前出现中间相。此外，中间相强烈地依赖于晶化温度。盐浓度高的样品表现出一个典型的转变温度Ttr，低于这个温度，溶液会变得浑浊。在Ttr附近结晶时，中间相由蛋白质簇组成；在Ttr以下，中间相对应于宏观的蛋白质聚集体，在结晶前可以进一步松弛成致密的液体相。然而，实验数据不能区分成核是发生在中间相内部还是外部。基于具有短程引力的胶体体系的平衡相行为，讨论了可能的情景。对所得晶体的结晶学分析表明，金属离子是晶格的组成部分。这两种类型的金属离子都可以通过桥联在晶格中产生新的蛋白质接触；然而，与锌相比，Y产生了更多的桥接接触。
Using bovine beta-lactoglobulin as a model system, we have studied the crystallization pathways in the presence of the di- and trivalent salts ZnCl2 and YCl3. Previous work has shown that protein solutions undergo a reentrant condensation (RC) phase behavior in the presence of YCl3, i.e., a condensed phase occurs in between two boundary salt concentrations, c*< c**. In the presence of ZnCl2, c* also exists, but protein solutions with high salt concentrations do not become completely clear at higher protein concentrations (>20 mg/mL). We thus denote this diffuse transition as pseudo-c**. Small angle X-ray scattering measurements show that the effective interactions change from repulsion to attraction near c*, and attractive interactions dominate around pseudoc**. Solutions near c* and pseudo-c** provide the optimized conditions for growth of high quality protein single crystals but with different pathways. While crystal growth near c* follows the classical one-step nucleation pathway, crystallization around pseudo-c** (for zinc) or c** (for yttrium) follows a nonclassical process with an intermediate phase appearing before crystallization. Furthermore, the intermediate phases strongly depend on the crystallization temperature. Samples with high salt concentrations exhibit a typical transition temperature, Ttr, below which the solutions become turbid. When crystallizing near Ttr, the intermediate phase consists of protein clusters; below Ttr, the intermediate phase corresponds to macroscopic protein aggregates which can further relax into a dense liquid phase before crystallization. However, the experimental data cannot distinguish whether nucleation occurs within or outside of the intermediate phase. Possible scenarios are discussed based on the equilibrium phase behavior of colloidal systems with a short-range attraction. The crystallographic analysis of the resulting crystals shows that metal ions are an integral part of the crystal lattice. Both types of metal ions can create new protein contacts in the crystal lattice via bridging; however, yttrium creates more bridging contacts compared to zinc.