Cherezov Vadim, Clogston Jeffrey, Papiz Miroslav Z, Caffrey Martin
College of Science and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland.
J Mol Biol. 2006 Apr 14;357(5):1605-18. doi: 10.1016/j.jmb.2006.01.049. Epub 2006 Feb 2.
The cubic phase or in meso crystallization method is responsible for almost 40 solved integral membrane protein structures. Most of these are small and compact proteins. A model for how crystals form by the in meso method has been proposed that invokes a transition between mesophases. In light of this model, we speculated that a more hydrated and open mesophase, of reduced interfacial curvature, would support facile crystallization of bigger and bulkier proteins. The proposal was explored here by performing crystallization in the presence of additives that swell the cubic phase. The additive concentration inducing swelling, as quantified by small-angle X-ray diffraction, coincided with a "crystallization window" in which two, very different transmembranal proteins produced crystals. That the swollen mesophase can grow structure-grade crystals was proven with one of these, the light-harvesting II complex. In most regards, the structural details of the corresponding complex resembled those of crystals grown by the conventional vapour diffusion method, with some important differences. In particular, packing density in the in meso-grown crystals was dramatically higher, more akin to that seen with water-soluble proteins, which accounts for their enhanced diffracting power. The layered and close in-plane packing observed has been rationalized in a model for nucleation and crystal growth by the in meso method that involves swollen mesophases. These results present a rational case for including mesophase-swelling additives in screens for in meso crystallogenesis. Their use will contribute to broadening the range of membrane proteins that yield to structure determination.
立方相或介晶结晶方法已解析了近40种完整的膜蛋白结构。其中大多数是小而紧凑的蛋白质。有人提出了一种通过介晶方法形成晶体的模型，该模型涉及中间相之间的转变。基于此模型，我们推测，一种水合度更高、界面曲率减小的开放中间相，将有助于更大、更庞大的蛋白质的轻松结晶。我们在此通过在存在使立方相膨胀的添加剂的情况下进行结晶来探索这一设想。通过小角X射线衍射定量的诱导膨胀的添加剂浓度，与一个“结晶窗口”相吻合，在这个窗口中，两种非常不同的跨膜蛋白产生了晶体。其中之一，光捕获II复合体，证明了膨胀的中间相可以生长出结构级别的晶体。在大多数方面，相应复合体的结构细节与通过传统气相扩散方法生长的晶体相似，但也存在一些重要差异。特别是，介晶生长的晶体中的堆积密度显著更高，更类似于水溶性蛋白质中的堆积密度，这解释了它们增强的衍射能力。通过涉及膨胀中间相的介晶方法进行成核和晶体生长的模型，对观察到的层状和紧密的面内堆积进行了合理化解释。这些结果为在介晶结晶形成筛选中加入中间相膨胀添加剂提供了合理的依据。它们的使用将有助于扩大能够进行结构测定的膜蛋白的范围。
