Development of high-dose time-resolved X-ray footprinting technologies to enable detailed structural and kinetics information to be obtained for challenging biological problems

摘要 这个项目的首要目标，无论是在资金奖励中还是在这次更新中，都是为了推动 X射线足迹质谱仪(XFMS)性能和可及性的结构生物学方法 以至于它成为全国生物医学研究人员的首选生物物理工具。XFMS 是一种溶液状态方法，用于在时间尺度上映射大分子中的溶剂可访问区域 微秒，产生关于构象、蛋白质-蛋白质动力学和结合水的信息 位置和动态。它已被用来获取各种范围内的有用结构信息 生物系统，从小的蛋白质到大的复合体，以及膜蛋白，以及 绘制抗体-靶标复合体中的相互作用区。作为最初奖项的一部分，我们制作了 通过开发独特的高通量和自动化技术，我们朝着主要目标取得了实质性进展 XFMS仪器，使全国的研究人员能够使用该方法。在这项建议的续期中，我们 计划在这一成功的基础上实施新的能力，以与赠款的原始目标保持一致。 具体地说，我们计划将荧光和拉曼光谱技术集成在一起，通过喷气输送实现快速混合 能力和尺寸排除直接与XFMS仪器联用。这些功能的整合 进入XFMS仪器的技术将使更具挑战性的生物系统成为 利用该方法进行了研究。虽然新的具体目标雄心勃勃，但它们自然建立在我们的 在开发复杂仪器和我们建立的成功研究团队方面有良好的记录 在第一次赠与期间。给出了这些技术的原理证明，以及 初步数据，该提案概述了涉及的重大技术挑战以及它们如何 都会被克服。由此产生的技术将是生物医学研究的重大收获 并将用于满足日益增长的使用XFMS方法的需求。

Abstract The overarching goal in this project, both in the funded award and in this renewal, is to advance the structural biology method of X-ray footprinting mass spectrometry (XFMS) in capability and accessibility such that it becomes a premiere biophysical tool for biomedical investigators around the country. XFMS is a solution state method used to map solvent accessible regions in macromolecules on a timescale of microseconds, yielding information on conformation, protein-protein dynamics, and bound water location and dynamics. It has been used to obtain useful structural information on a diverse range biological systems, from small proteins to large complexes, as well as membrane proteins, and for mapping interaction regions in antibody-target complexes. As part of the original award, we made substantial progress towards our main goal by developing a unique high-throughput and automated XFMS instrument, enabling use of the method to researchers nationwide. In this proposed renewal, we plan to build on this success to implement new capabilities in keeping with the original goal of the grant. Specifically, we plan to integrate fluorescence and Raman spectroscopies, fast mixing with jet delivery capability, and size exclusion directly inline with the XFMS instrument. The integration of these technologies into the XFMS instrument will enable even more challenging biological systems to be studied using the method. While the new specific aims are ambitious, they build naturally from our proven track record in developing complex instrumentation and the successful research team we built during the first grant period. Proof of principle for these technologies is presented, along with preliminary data, and the proposal outlines the significant technical challenges involved and how they will be overcome. The resulting technologies will be a significant gain to the biomedical research community and will be used to meet the increasing demand for access to the XFMS method.