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

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.