Membrane proteins and their complexes act as cellular gatekeepers, regulating the traffic of critical chemical agents and acting as highly-selective extracellular receptors. As such, membrane proteins are amongst the most promising potential therapeutic targets for a host of diseases, including many cancers. The refractory nature of many membrane proteins to widely- used technologies such as NMR and X-ray crystallography has resulted in structural information on these critical molecular machines lagging significantly behind our knowledge of soluble proteins. Membrane protein structure is often highly dependent on the local membranous environment, which often generates an insurmountable level of chemical noise for most structural probes. Chemical cross-linking mass spectrometry (CXL-MS) and Ion Mobility-Mass Spectrometry (IM-MS) have emerged as technologies capable of filling our current gap in knowledge of membrane protein complex structure and topology when combined with modeling techniques. Recent reports have demonstrated how distance and contact constraints derived from CXL-MS and IM-MS measurements can be used to deduce topologies for highly-complex macromolecular membrane protein machines that have frustrated all other attempts at detailed structural characterization. Expanding the use of these approaches beyond a few examples and capitalizing on the recent promise demonstrated by MS technologies for membrane protein structure determination will require overcoming several key challenges. We propose to optimize new structural mass spectrometry technologies for mitochondrial membrane protein complexes. We will use the model mitochondrial protein TspO to build our approach, and will also study the influence of membrane and detergent conditions on its structure. The structural constraints obtained from IM and CXL-MS will be used to build more accurate models of TspO and its multimers, as well as define the protein and lipid stoichiometries for the system. We will then extend our technologies to discover the structure of several mitochondrial membrane protein complexes, important in cancer etiology.

Public Health Relevance

Membrane proteins and their complexes have many critical functions, including regulating the traffic of critical chemical agents, acting as highly-selective extracellular receptors, and maintaining cell-cell interactions. As such, membrane proteins are amongst the most promising therapeutic targets for a host of diseases, including many cancers, but they are refractory to the most widely-used tools in structural biology. This situation has resulted in a relatively small amount of structural information for these critical molecular machines when compared to our knowledge of soluble proteins. This project targets the development and application of new structural mass spectrometry technologies capable of elucidating the structures and dynamics of multi-component membrane protein complexes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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Hagen, Susan E; Liu, Kun; Jin, Yafei et al. (2018) Synthesis of CID-cleavable protein crosslinking agents containing quaternary amines for structural mass spectrometry. Org Biomol Chem 16:8245-8248
Zhang, Yang; Halder, Sabyasachi; Kerr, Richard A et al. (2016) Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-?K. J Biol Chem 291:10347-62