Innovative Native Ion Mobility Approaches for Transformational Measurements in Structural Biology

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Project Summary Advancements in biophysical techniques, such as X-ray and cryoEM, have undoubtedly accelerated determination of protein structure. However, it still remains challenging to capture snapshots of protein folding intermediates, including non-native states, and breathing motions that protein assemblies undergo to perform their biological function. Moreover, understanding how molecules, such as lipids, modulate protein structure and function is of paramount biological importance. Over the past two decades, mass spectrometry (MS) of intact protein complexes, often referred to as native MS, has emerged as an indispensable biophysical technique whereby non-covalent interactions and protein structure are preserved within the mass spectrometer. Native MS is a rapid and sensitive technique that has already provided invaluable information on subunit stoichiometry and topology, allostery and cooperativity for individual ligand binding events, including their binding thermodynamics. The coupling with ion mobility (IM), a separation technique based on molecule charge and shape, further enhances the capabilities of native MS where it has enabled collision cross section (CCS) measurements for large protein complexes, identification of different conformations for peptides and stabilizing ligands using collision induced unfolding, and insight in folded and denatured structure(s) of proteins. However, low- resolution commercial IM-MS instrumentation has not changed since its introduction 12 years ago. Herein, this proposal seeks to develop transformative native IM-MS technologies with high-resolution IM and MS capabilities that can address modern questions in structural biology, such as conformational dynamics, including those that may have remained “hidden”, within membrane transporters under turnover conditions. In order to achieve these transformative goals, an interdisciplinary team of researchers whose expertise spans the fields of protein biophysics, expression and purification of proteins inclusive of membrane proteins, as well as traditional protein structure characterization, such as X-ray crystallography, has been assembled. Team members also possess decades of experience in the field of mass spectrometry inclusive of fundamental ion chemistry/physics, seminal contributions that have spawned MS proteomics, and related areas of analytical mass spectrometry and ion mobility- mass spectrometry. Collectively, the background and expertise of this research team is uniquely positioned to transform the field of IM-MS in the area of structural biology. In short, the proposed transformative research will lead to forefront IM-MS instrumentation that is poised to provide unprecedented insights into the structure and assembly of protein complexes and push the field into new frontiers of research.