Mapping the Crystallization Slot for Membrane Prote(RMI)

描述(申请人提供)：这项建议旨在开发一种基于自作用层析(SIC)的新技术来测量膜蛋白之间的相互作用，并将该技术应用于制备这些分子的有序晶体。这项工作是长期努力的一部分，目的是开发有助于获取膜蛋白结构信息的方法。 完整的膜蛋白介导了活细胞进行的一些最重要的过程，约占基因组编码蛋白质的30%。然而，它们只占已知结构蛋白质的不到1%，这主要是因为它们在获得衍射性晶体方面造成了极大的困难。因此，能够使完整的膜蛋白结晶合理化和简化的技术和方法将对结构生物学产生巨大的好处。 第二维里系数B22提供了“结晶性”的一个可量化的量度，应该可以利用这个量来合理地筛选和优化膜蛋白结晶的条件。然而，用于测量B22的标准技术不适用于膜蛋白的高通量测量。SIC提供了一种快速有效地测量膜蛋白B22的方法，绕过了与标准技术相关的问题。 该项目的具体目标是优化碳化硅技术在整合膜蛋白中的应用，并使用碳化硅获得的维里系数测量来检验关于膜蛋白结晶的最佳方法的假设，并最终快速识别和优化这些分子的结晶条件。 膜蛋白在维持细胞正常功能和许多病理过程中都起着至关重要的作用。它们是大量药物和毒素的靶标，也介导了对许多其他药物的耐药性。对于深入了解这些分子的基本功能，以及发现和开发新的治疗剂，高分辨率的结构信息是绝对必要的。

DESCRIPTION (provided by applicant): This proposal aims to develop a novel technology based upon self-interaction chromatography (SIC) to measure interactions between membrane proteins, and to apply this technology to the preparation of well-ordered crystals of these molecules. This work is part of a long-term effort to develop methods that will facilitate the acquisition of structural information for membrane proteins. Integral membrane proteins mediate some of the most important processes carried out by living cells, and represent approximately 30% of the proteins encoded in the genome. However, they represent fewer than 1 % of the proteins of known structure, largely because of the extreme difficulties they pose in obtaining diffraction quality crystals. Technologies and methodologies that could rationalize and simplify the crystallization of integral membrane proteins would therefore represent a huge boon to structural biology. The second virial coefficient B22 provides a quantifiable measure of "crystallizability," and it should be possible to exploit this quantity to rationally screen and optimize conditions for membrane protein crystallization. However, standard technologies for measuring B22 are not adaptable to high-throughput measurements with membrane proteins. SIC offers a means for rapid and efficient B22 measurements of membrane proteins that circumvents the problems associated with the standard technologies. The specific aims of this project are to optimize the application of SIC technology to integral membrane proteins, and to use virial coefficient measurements obtained by SIC to test hypotheses about the best ways to crystallize membrane proteins and, ultimately, to quickly identify and optimize crystallization conditions for these molecules. Membrane proteins play crucial roles in both maintaining normal cellular function and in a large number of pathologies. They are the targets of huge number of drugs and toxins, and also mediate resistance to many other drugs. High resolution structural information is absolutely required for any in-depth understanding of the basic functioning of these molecules, as well as for the discovery and development of new therapeutic agents.