Membrane protein crystallization
Membrane protein crystallization
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DOI：
10.1016/s1047-8477(03)00043-1
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发表时间：
2003-04-01
影响因子：
3
通讯作者：
Caffrey, M
中科院分区：
文献类型：
--
作者：
Caffrey, M
对膜蛋白的高分辨率结构信息的需求正在迫在眉睫并且在不断增长。目前，获得它的唯一可靠的方法是结晶学。从蛋白质到结构的限速步骤是晶体的产生。综述了目前膜蛋白结晶领域中流行的思想和实验方法。已建立已久的基于表面活性剂的方法已被广泛回顾，在此不作详细介绍。相反，重点放在最新的方法上，所有这些方法都利用了脂类和洗涤剂的自发自组装特性，如囊泡(囊泡融合法)、盘状胶束(双胶束法)和液晶或中间相(介相或立方相方法)。相信基本的相科学知识对于理解这些分类结晶策略的分子基础是不可或缺的，本文从关于脂类、中间相和相科学的简要入门开始，并讨论了应用于脂类的形式和功能的相关问题。研究了与获得足够量的均一膜蛋白或其部分相关的实验挑战和解决方案。从以下几个角度描述了三次相法：它在实践中是如何进行的，它的普遍适用性和迄今取得的成功，以及该过程所必需的中间相的性质。该方法的实际方面包括盐、洗涤剂和筛分溶液效果；低温结晶；根据目标蛋白质定制立方相；不同的立方相类型；处理低蛋白样品、无色蛋白质、微晶体和辐射损伤；立方相内的传输用于药物设计、辅因子保留和相变；使用光谱学进行质量控制；收获晶体；以及用于高通量筛选的小型化和自动化。本部分最后提出了膜蛋白晶体在介观体系中成核和生长的假说。到目前为止，双胞体和囊泡融合法已经产生了一种膜蛋白--细菌视紫红质的晶体。回顾了这两种方法的实验细节，并对它们在未来的普遍适用性进行了评论。通过类比微重力下的结晶和外延，这三种新方法都是合理的。在膜蛋白晶体发生领域的网络资源的列表被包括在内。(C)2003年埃尔塞维尔科学公司(美国)。版权所有。
The need for high-resolution structure information on membrane proteins is immediate and growing. Currently, the only reliable way to get it is crystallographically. The rate-limiting step from protein to structure is crystal production. An overview of the current ideas and experimental approaches prevailing in the area of membrane protein crystallization is presented. The long-established surfactant-based method has been reviewed extensively and is not examined in detail here. The focus instead is on the latest methods, all of which exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method). In the belief that a knowledge of the underlying phase science is integral to understanding the molecular basis of these assorted crystallization strategies, the article begins with a brief primer on lipids, mesophases, and phase science, and the related issue of form and function as applied to lipids is addressed. The experimental challenges associated with and the solutions for procuring adequate amounts of homogeneous membrane proteins, or parts thereof, are examined. The cubic-phase method is described from the following perspectives: how it is done in practice, its general applicability and successes to date, and the nature of the mesophases integral to the process. Practical aspects of the method are examined with regard to salt, detergent, and screen solution effects; crystallization at low temperatures; tailoring the cubic phase to suit the target protein; different cubic-phase types; dealing with low-protein samples, colorless proteins, microcrystals, and radiation damage; transport within the cubic phase for drug design, cofactor retention, and phasing; using spectroscopy for quality control; harvesting crystals; and miniaturization and robotization for high-throughput screening. The section ends with a hypothesis for nucleation and growth of membrane protein crystals in meso. Thus far, the bicelle and vesicle-fusion methods have produced crystals of one membrane protein, bacteriorhodopsin. The experimental details of both methods are reviewed and their general applicability in the future is commented on. The three new methods are rationalized by analogy to crystallization in microgravity and with respect to epitaxy. A list of Web resources in the area of membrane protein crystallogenesis is included. (C) 2003 Elsevier Science (USA). All rights reserved.