Strategies for crystallizing membrane proteins
Strategies for crystallizing membrane proteins
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DOI：
10.1007/bf02150674
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发表时间：
1996-02-01
影响因子：
3
通讯作者：
Loll, PJ
中科院分区：
文献类型：
--
作者：
Garavito, RM;Picot, D;Loll, PJ
尽管通过 X 射线晶体学解析的膜蛋白结构数量不断增加，但膜蛋白的结晶仍然是一项具有挑战性的工作。决定结晶实验成功的关键因素是膜蛋白样品的纯化和制备。此外，还有一个额外的复杂性，即必须优化结晶条件，以便在去垢剂存在下使用，尽管用于结晶大多数膜蛋白的方法本质上是可溶性蛋白质结晶标准方法的直接应用。在结晶试验之前和期间，需要了解和控制去污剂在膜蛋白的稳定性和聚集以及蛋白质-去污剂复合物的胶体特性中所起的作用。所有 X 射线质量的膜蛋白晶体都是从去污剂溶解的蛋白质制剂中生长出来的，其中来自膜的异质天然脂质已被均质的去污剂环境所取代。正是这种单分散、各向同性的膜蛋白溶液的制备使得常规用于可溶性蛋白的标准结晶方法得以成功应用。在这篇综述中，解决了蛋白质纯化和样品制备的问题以及膜蛋白结晶方法的新改进。还讨论了胶束或蛋白质洗涤剂聚集体形式的洗涤剂的物理行为如何影响结晶以及已发表的方案对新膜蛋白系统的适应。总的结论是，如果可以在合适的去污剂系统中制备纯的单分散制剂，则许多完整的膜蛋白可以结晶。
Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in the stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlled before and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by a homogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.