Within each organism proteins are at work carrying out activities which impact every aspect of cellular function, from replication to cell death. A ke factor in achieving both a wide range of functions and high degrees of efficiencies is the ability of proteins to self-assemble into macromolecular `machines'. The 3D structures of these assemblies are crucial for understanding normal and disease states and for drug development. However, most structures remain unknown and are refractory to current technologies. Current approaches to this challenge mainly rely on the direct conversion of isolated protein complexes into structures of atomic-detail via high-throughput X-ray or NMR technologies. While highly successful, these methods require pure samples in large quantities, typically optimized to either generate crystals or remove spectral background. Furthermore, transient and polydisperse assemblies that exist within complex mixtures cannot be analyzed. Alternative methodologies such as electron microscopy (EM) and small angle X-ray scattering (SAXS) allow determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited, in general, to homogeneous complexes. Consequently there is a need to develop new approaches that define subunit stoichiometry, composition, interface structure, shape, and the interaction dynamics of heterogeneous macromolecular complexes of clear biomedical importance. This proposal renewal seeks to construct new, innovative structural mass spectrometry techniques that 1) leverage controlled protein complex disruption and native mass spectrometry to build detailed models of protein-protein interfaces 2) validate recent observations linking gas-phase protein unfolding to solution-phase protein domain structure in order to create a new tool for protein subunit model construction 3) enhance chemical cross-linking reagents to increase information content and quantify changes in protein interactions and conformations in vitro and in vivo 4) build new protein modeling tools capable of integrating multiple sources of structural mass spectrometry data and 5) create new chemical reagents that enable the comprehensive top-down sequencing, or enhanced stability, of megadalton-scale intact protein complexes. All of this technology will be brought to bear to discover the structures of a series of selected protein complexes, each having a critical link to human disease.
Living cells are dependent on molecular machines composed of protein subunits for critical biological functions. Knowledge of their structures allows understanding of their functions in normal and diseased organisms, thus playing a central role in drug development. How the subunits combine to form this very complex machinery has been a central goal in biomedical research, and traditional X-ray and NMR methods have made great progress, but many proteins remain refractory to such technologies, resulting in a plethora of protein complexes having unknown structures. Here, we propose to develop a suite of new structural mass spectrometry strategies for analysis of protein complexes based on a combination of ion mobility, and chemical crosslinking, native mass spectrometry, and computational modeling. When integrated with radical labeling and hydrogen-deuterium exchange, such structural mass spectrometry has the ability to capture structure information from small amounts of unlabeled protein, present within complex mixtures that remain difficult or impossible to analyze using classical structural biology probes.
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