An instrument to measure protein amide H/D exchange within a second

描述（由申请人提供）：将组装一台能够在规定温度下在一秒内测量H/D交换的仪器，以测试可以在显着更高的时间分辨率下获得关于蛋白质构象变化的定量数据的假设。这将通过开发一种新型快速在线混合模块来实现，该模块能够在1秒后确定氘掺入水平，而不是目前的30秒测量能力。使用提议的仪器收集的定量数据对于确定蛋白质结构中的哪些变化调节信号通路，例如由激酶和核受体控制的信号通路，将特别有用。初步结果表明，从长远来看，从拟议仪器中获得的数据将简化和加速新药的发现，具有更大的特异性，从而为患有慢性疾病（如糖尿病、动脉粥样硬化和与癌症相关的急性疾病）的患者提供更个性化的治疗。

DESCRIPTION (provided by applicant): An instrument capable of measuring H/D exchange within a single second at a defined temperature will be assembled to test the hypothesis that quantitative data could be obtained about protein conformational changes at a significantly higher temporal resolution. This will be accomplished by development of a novel rapid in-line mixing module capable of determining the deuterium incorporation level after 1 second, in place of the current measuring capability of 30 seconds. The quantitative data collected using the proposed instrument will be especially powerful for determining what changes in protein structure regulate signaling pathways, such as those controlled by kinases and nuclear receptors. Preliminary results indicate that data obtained from the proposed instrument will in the long term simplify and speed discovery of new medicines with greater specificity to allow more individualized treatments for patients suffering from chronic diseases like diabetes, atherosclerosis, and acute conditions related to cancer. Specific Aims Phase I of the project encompasses three Specific Aims over a period of 18 months whereupon a clearly defined end-point Milestone represents a significant achievement. In Specific Aim #1, equipment to achieve rapid mixing for both initiation and quench of the exchange reaction will be assembled and tested. In Specific Aim #2 the assembled rapid mixing module along with means to control temperature, will be swapped for the 30 sec mixing module of the current H/D exchange system. Finally in Specific Aim #3 the newly assembled instrument, demonstrated to completely mix solutions in one second, will be tested with proteins and protein:ligand complexes with known H/D exchange properties. Phase I end point Milestone: Determine H/D Exchange within 1 sec at a defined temperature Advancement of the project is dependent upon demonstrating determination of H/D exchange within one second using the temperature-controlled rapid mixing system. The commercialization of such an instrument, as proposed in Phase II, will provide a way to more accurately define protein dynamics and protein:protein interactions as needed for drug discovery, basic science, and proteomics research. As the test molecule we chose a representative of the nuclear receptor (NR) family that regulates pathways involved in chronic diseases such as diabetes, cancer and atherosclerosis, and is the target of drugs exhibiting annual sales nearing $13B.