Grant: Research Tools and Instruments

This proposal requests funding to purchase a state-of-the-art system for separating biologically intact macromolecular complexes and elucidating their oligomeric states, stoichiometries and polydispersities. The complexes are derived from natural sources including raw membrane from plants, fungi and animal sources, whole cells and tissues, and consist of multiple proteins, lipid species and amphipathic polymers which have been designed for spontaneous release into nanoparticles between 5 and 20 nm. This overcomes the limitations of conventional detergents and allows unstable, labile and rare complexes to be produced cost effectively and studied in native states, and offers a step-change for molecular and structural biology research as well as applications in biotechnology, defense, energy and pharmaceutical industries. We will purchase and install Canada's first membrane nanodisc separation system with multi-angle static light scattering (SEC-MALS), allowing generation of pure, stable and active assemblies without harsh conditions or detergents. A range of sizes of the nanodiscs can be selected based on the polymer and extraction method used, and several matrices can be used for clean separation of the macromolecular assemblies. The polydispersities are measured by the SEC-MALS systems, and homo-oligomeric and hetero-oligomeric states can be detected. These states are particularly relevant for channels, lipid biosensors and complex molecular machines that cannot be reconstituted from recombinant sources or produced by any other means. This SEC-MALS system is needed for by a growing collaborative community of membrane biochemists, structural biologists and enzymologists at the University of Alberta and beyond, and will enable the study of important targets from diverse prokaryotic pathogens, prions, and eukaryotic species as well as discovery of specific ligands and probe molecules. The co-applicants are members of an NSERC CREATE training program focussed on membrane proteins, and lead an international network leading the development of native nanodiscs. The Department of Biochemistry actively maintains and develops infrastructure for structural biology, and will ensure that the system forms a well-run and supported research resource. the application outline the research programs of HQP who will use the requested system including four co-located labs in the Department of Biochemistry. In fact all 8 NSERC-funded labs in the Department of Biochemistry and Membrane Protein Training Group fully support this equipment request and could provide further details of how their research programs would also benefit significantly from the access provided. We have only focussed on four major users here, but are committed to running an open resource similar to NANUC, which has offered accessible NMR services and training to national and international users for over 15 years.