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.

整合膜蛋白的正常功能是许多基本细胞功能所必需的，此类蛋白质构成了大多数药物靶点。尽管膜蛋白对人类健康具有明显的重要性，但我们对其结构、功能和动力学的认识和理解远远落后于可溶性蛋白。尽管过度表达、稳定、纯化、重构和表征带来了挑战，但有序的膜蛋白单晶的生长仍然是膜蛋白结构测定的最重要瓶颈。膜蛋白结晶的问题与膜蛋白的结晶有直接关系。蛋白质驻留在脂质双层中，并且稳定且具有功能，而生物化学、分子生物学和生物物理研究以及结晶实验是在水溶液中进行的。洗涤剂具有复杂的行为，形成许多蛋白质-洗涤剂和洗涤剂-洗涤剂相，这些相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它们的溶解度很大程度上取决于洗涤剂相行为的物理化学性质。在这个项目中，我们提出了一种膜蛋白结晶策略，该策略将消除与蛋白质-洗涤剂复合物相关的问题。我们将制定一种策略来识别富含过度表达的目标膜蛋白的红细菌膜组分，然后将这些膜直接掺入脂质立方材料中进行结晶，从而避免需要洗涤剂的溶解和纯化步骤。脂质立方基质应提供成功进行结晶试验所需的结构稳定性和动态灵活性。实验最初将使用一些典型的测试膜蛋白进行，然后是在红细菌中表达并由膜蛋白生产核心设施提供的目标蛋白。