Method development to investigate the assembly of a ternary holoreceptor complex**

这项拨款提案包括建立一种方法，在体外量化信号蛋白与受体和辅助受体组合之间的结合亲和力。Paraza Pharma是一家研发生物技术应用的综合性研究机构。该公司的目标是开发新的方法来纯化分泌的和膜完整的蛋白质，这些蛋白质有助于细胞表面的信号和细胞通讯；该项目的另一个方面旨在阐明当信号蛋白与其假定的受体/辅助受体复合体结合时，从细胞外环境到细胞质的全受体转导信号的形成。了解信号蛋白如何与由受体和辅助受体组合组成的全受体复合体结合仍然是Paraza Pharma提出的一个基本问题和技术挑战。这一项目需要高灵敏度的生物物理方法来测量信号通路中不同蛋白质组分之间的结合亲和力，还需要膜蛋白领域的专业知识，膜蛋白因其内在的不稳定性和体外操作的难度而被认为是具有挑战性的样品。卡尔梅特斯博士是膜蛋白特性和大分子结晶学方面的专家，他将为Paraza Pharma开发一种方法来生产和纯化膜信号转导蛋白，并量化它们与同源信号蛋白的结合亲和力。将开发的实验设计与加拿大的工业和加拿大知识的进步高度相关；预计它将为加拿大生物技术、生物科学和生物工业部门未来的应用开发开辟新的途径。

The grant proposal consists in establishing a method to quantify binding affinities between a signaling protein and a combination of receptors and coreceptors in vitro. Paraza Pharma is a fully integrated research organization developing biotechnological application. The company aims to develop novel methods for the purification of secreted and membrane integral proteins that contribute to signaling and cellular communication at the cell surface; another aspect of the project aims at elucidating the formation of an holoreceptor transducing signal from the extracellular milieu to the cytoplasm upon binding of a signaling protein with its putative receptor/coreceptor complex. Understanding how a signaling protein binds to a holoreceptor complex composed of a combination of receptors and coreceptors remains a fundamental question and a technical challenge raised by Paraza Pharma. Such project requires highly sensitive biophysical methods to measure binding affinities between the diverse protein components of the signaling pathway, as well as it requires specialized expertise in the field of membrane proteins, which are considered challenging samples due to their intrinsic instability and difficulty to manipulate in vitro. Dr. Calmettes, an expert in membrane protein characterization and macromolecular crystallography, will develop for Paraza Pharma a method to produce and purify membrane-signal-transducing proteins and to quantify their binding affinities with a cognate signaling protein. The experimental design to be developed is highly relevant for the Canadian industry and the advancement of knowledge in Canada; it is anticipated to open new avenues for the development of future applications in biotechnology, bioscience and bio-industrial sectors in Canada.