Integrated In-membrane Crystallization

许多基本的细胞功能都需要膜蛋白的适当功能，这类蛋白质构成了大多数药物靶点。尽管膜蛋白对人类健康的重要性显而易见，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。尽管在过表达、稳定化、纯化、重构和表征方面存在挑战，但有序膜蛋白单晶的生长仍然是膜蛋白结构测定的最重要瓶颈。膜蛋白结晶中的问题直接与膜蛋白的结晶性有关。 蛋白质存在于双分子层中，并且在双分子层中是稳定和功能性的，而生物化学、分子生物学和生物物理学研究以及结晶实验在水溶液中进行。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（例如pH、离子强度、粘度、温度等）的影响。此外，膜蛋白的结构和功能完整性及其溶解度强烈地依赖于洗涤剂相行为的物理化学性质。在这个项目中，我们提出了一个策略，膜蛋白的结晶，将消除与蛋白质洗涤剂复合物的问题。我们将开发一种策略，以确定红细菌属膜组分，富含过表达的目标膜蛋白，然后将这些膜直接纳入结晶的立方晶系材料，避免需要溶解和纯化步骤，需要洗涤剂。所述双曲晶系立方基质应提供进行成功的结晶试验所必需的结构稳定性和动态灵活性。实验将首先用许多典型的测试膜蛋白进行，然后是将在红细菌中表达并由膜蛋白生产核心设施提供的靶蛋白。

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.