Caffrey Martin
Biochemistry, Biophysics, Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210-1106, USA.
J Struct Biol. 2003 Apr;142(1):108-32. doi: 10.1016/s1047-8477(03)00043-1.
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.
对于膜蛋白高分辨率结构信息的需求迫切且不断增长。目前，获取此类信息的唯一可靠方法是通过晶体学手段。从蛋白质到其结构的限速步骤是晶体的产生。本文概述了膜蛋白结晶领域当前流行的观点和实验方法。基于表面活性剂的长期方法已被广泛综述，在此不再详细探讨。相反，重点在于最新方法，所有这些方法都利用了脂质和去污剂作为囊泡（囊泡融合法）、盘状胶束（双分子层方法）以及液晶或中间相（在中间相或立方相方法）的自发自组装特性。鉴于了解基础相科学对于理解这些不同结晶策略的分子基础不可或缺，本文开篇简要介绍了脂质、中间相和相科学，并探讨了与脂质相关的形式和功能问题。研究了获取足够量均匀膜蛋白或其部分所面临的实验挑战及解决方案。从以下几个方面描述立方相方法：实际操作过程、其目前的普遍适用性和成功案例，以及该过程中不可或缺的中间相的性质。还考察了该方法在盐、去污剂和筛选溶液影响方面的实际问题；低温结晶；使立方相适合目标蛋白；不同的立方相类型；处理低蛋白样品、无色蛋白、微晶和辐射损伤；用于药物设计、辅因子保留和相位测定的立方相内转运；利用光谱进行质量控制；收获晶体；以及用于高通量筛选的小型化和自动化。该部分最后提出了关于中间相膜蛋白晶体成核和生长的假设。到目前为止，双分子层和囊泡融合方法仅产生了一种膜蛋白细菌视紫红质的晶体。本文回顾了这两种方法的实验细节，并对它们未来的普遍适用性进行了评论。通过与微重力结晶和外延进行类比，对这三种新方法进行了合理说明。本文还列出了膜蛋白晶体生成领域的网络资源清单。
