Integrated In-membrane Crystallization

完整的膜蛋白的正确功能是许多基本细胞功能所必需的，而这类蛋白构成了大多数药物靶点。尽管膜蛋白对人类健康有明显的意义，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。尽管膜蛋白的过度表达、稳定、纯化、重组和鉴定都面临着挑战，但有序的膜蛋白单晶的生长仍然是膜蛋白结构确定的最大瓶颈。膜蛋白结晶过程中的问题直接与膜 蛋白质存在于脂类双层中，并且具有稳定性和功能性，而生化、分子生物学和生物物理研究以及结晶实验则在水溶液中进行。洗涤剂具有复杂的行为，形成大量的蛋白质-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的完整性以及它们的溶解性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，我们提出了一种膜蛋白结晶的策略，该策略将消除与蛋白质-洗涤剂复合体相关的问题。我们将开发一种策略来鉴定富含过表达的目标膜蛋白的红细菌膜组分，然后将这些膜直接结合到脂质立方体材料中进行结晶，从而绕过需要洗涤剂的增溶和纯化步骤。脂质立方体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。实验将首先对一些具有聚集性的膜蛋白进行，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目标蛋白。

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.