Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation.
Crystallization of transmembrane proteins in cubo: mechanisms of crystal growth and defect formation.
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DOI：
10.1016/j.jmb.2004.09.022
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发表时间：
2004-11
影响因子：
5.6
通讯作者：
Yasser Qutub;I. Reviakine;C. Maxwell;Javier Navarro;E. Landau;P. Vekilov
中科院分区：
文献类型：
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作者：
Yasser Qutub;I. Reviakine;C. Maxwell;Javier Navarro;E. Landau;P. Vekilov
膜蛋白的结晶是阐明其结构和理解其功能的主要绊脚石。膜蛋白质结晶的新概念，从cubo立方相，“在立方”，已经产生了良好的有序晶体和高分辨率的结构的几个膜蛋白质，但进展缓慢，由于缺乏了解的分子机制的蛋白质运输，晶体成核，生长，和缺陷形成立方。在这里，我们研究在分子和介观分辨率与原子力显微镜的形态在立方生长的细菌视紫红质晶体在惰性缓冲液和洗涤剂蚀刻过程中。结果表明，晶体成核发生后，高度弯曲的立方相的局部重排成层状结构，这是类似于原生膜。晶体在被这种层状结构包围的体立方相内生长，由此朝向生长的晶体层的传输被约束在单个薄片内。这种机制导致缺乏位错，在许多位置产生新的晶体层，并导致空隙和块边界。这些缺陷的特征宏观长度尺度表明，晶体生长的附着单分子的核。这些见解的成核，生长和运输的机制，在立方膜蛋白结晶的合理设计提供了指导，并承诺进一步推进该领域。
Crystallization of membrane proteins is a major stumbling block en route to elucidating their structure and understanding their function. The novel concept of membrane protein crystallization from lipidic cubic phases, “in cubo”, has yielded well-ordered crystals and high-resolution structures of several membrane proteins, yet progress has been slow due to the lack of understanding of the molecular mechanisms of protein transport, crystal nucleation, growth, and defect formation in cubo. Here, we examine at molecular and mesoscopic resolution with atomic force microscopy the morphology of in cubo grown bacteriorhodopsin crystals in inert buffers and during etching by detergent. The results reveal that crystal nucleation occurs following local rearrangement of the highly curved lipidic cubic phase into a lamellar structure, which is akin to that of the native membrane. Crystals grow within the bulk cubic phase surrounded by such lamellar structures, whereby transport towards a growing crystalline layer is constrained to within an individual lamella. This mechanism leads to lack of dislocations, generation of new crystalline layers at numerous locations, and to voids and block boundaries. The characteristic macroscopic lengthscale of these defects suggests that the crystals grow by attachment of single molecules to the nuclei. These insights into the mechanisms of nucleation, growth and transport in cubo provide guidance en route to a rational design of membrane protein crystallization, and promise to further advance the field.