Integrated In-membrane Crystallization

集成膜内结晶

基本信息

批准号：
7138479
负责人：
PHILIP D LAIBLE
金额：
$ 20.51万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-23 至 2010-07-31
项目状态：
已结题

项目摘要

The proper function of integral membrane proteins is required for a number of essential cellular functions, and this class of proteins comprises the majority of drug targets. Despite the obvious significance of membrane proteins to matters of human health, our knowledge and understanding of their structure, function, and dynamics lags far behind that of soluble proteins. Although challenges are presented by overexpression, stabilization, purification, reconstitution and characterization, the growth of well-ordered single crystals of membrane proteins is still the most significant bottleneck for the structure determination of membrane proteins. The problems in the crystallization of membrane proteins are directly related to the fact that membrane proteins reside, and are stable and functional in a lipidic bilayer, whereas biochemical, molecular biological and biophysical studies and crystallization experiments are conducted in aqueous solution. Detergents have a complex behavior, forming numerous protein-detergent and detergent-detergent phases which are influenced by the physical chemical parameters used for crystallization such as the pH, ionic strength, viscosity, temperature etc. Furthermore, the structural and functional integrity of membrane proteins and their solubility strongly depend on the physical chemical properties of the phase behavior of the detergent. In this project, we propose a strategy for crystallization of membrane proteins that will eliminate the problems associated with protein-detergent complexes. We will develop a strategy to identify Rhodobacter membrane fractions that are enriched in overexpressed target membrane protein and will then incorporate these membranes directly into lipidic cubic materials for crystallization, circumventing the need for solubilization and purification steps that require detergents. The lipidic cubic matrices should provide the structural stabilization and dynamic flexibility necessary to conduct successful crystallization trials. Experiments will be carried out initially with a number of paradigmatic test membrane proteins, to be followed by the target proteins that will be expressed in Rhodobacter and provided by the Membrane Protein Production core facility.

积分膜蛋白的正确功能是许多必需细胞功能所需的，并且这类蛋白质包括大多数药物靶标。尽管膜蛋白对人类健康问题具有明显的意义，但我们对它们的结构，功能和动力学的了解和理解远远落后于可溶性蛋白。尽管通过过表达，稳定，纯化，重构和表征提出了挑战，但膜蛋白有序的单晶的生长仍然是膜蛋白结构测定的最显着瓶颈。膜蛋白结晶的问题与膜直接相关蛋白质驻留在脂质双层中，并且在水溶液中进行生化，分子生物学和生物物理研究以及结晶实验，并且在脂质双层中具有稳定且功能性。洗涤剂具有复杂的行为，形成了许多蛋白质含量和洗涤剂含量的相位，它们受到用于结晶的物理化学参数的影响，例如pH，离子强度，粘度，温度等。此外，膜蛋白的结构和功能完整性以及它们的溶解性强烈地依赖于阶段的物理化学性质。在这个项目中，我们提出了一种结晶膜蛋白的策略，该策略将消除与蛋白质含量复合物相关的问题。我们将制定一种策略来鉴定富含过表达的靶膜蛋白的杜鹃花膜级分，然后将这些膜直接掺入脂质立方材料中，以进行结晶，从而避免了需要溶解和纯化步骤的需求，这些步骤需要清洁。脂质立方矩阵应提供成功结晶试验所需的结构稳定和动态灵活性。实验最初将使用许多范式测试膜蛋白进行，其次是靶蛋白，该靶蛋白将在杜鹃杆菌中表达，并由膜蛋白生产核心设施提供。