EAGER: Towards DNA-based Tools for Single-particle Cryo-electron Microscopy of Membrane Proteins

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This research will develop molecular tools that allow the study of membrane proteins (MPs), which are among the most important, but least understood components of cells. All cells are surrounded by lipid membranes that are almost impermeable to water, salts or nutrients that cells need. For this reason, many membrane proteins (MPs) are inserted into the membranes that control cellular functions such as material transport, sensing, intercellular communication, cell adhesion, and energy conversion. MPs are also the targets for many therapeutic drug molecules. Knowledge of the molecular structure of MPs is necessary to understand the underlying molecular mechanisms of their function and can guide the development of therapeutic drugs for many common diseases. However, MPs are difficult to study and therefore the molecular structure of most MPs is still unknown. The goal of this project is to develop broadly applicable new tools using DNA nanotechnology that will facilitate solving MP structures with cryo-electron microscopes. This project will provide research opportunities for graduate, undergraduate and high school students from underrepresented minority groups through several established programs, which will inform them about experimental research and potential career opportunities. Finally, introductory physics courses will be strengthened by developing modules that underline interdisciplinary aspects and applications of physical principles, and an interdisciplinary molecular biophysics course will be developed. These efforts will train the next generation of diverse, interdisciplinary leaders in STEM research and technology.Single-particle cryo-electron microscopy (cryo-EM) is becoming the standard method for MP structure determination, but several experimental challenges have prevented solving more than 1% of human MP structures so far. The overall goal of this project is to establish DNA-lipid nanodiscs (DLNs) as a radically new customizable nanoscale lipid bilayer mimetic for single-particle cryo-EM of MPs. This DNA nanotechnology-based approach will overcome existing limitations of established bilayer mimetics and offer unprecedented control over structural, chemical and physical design parameters that could transform MP research. Such a paradigm shift involves significant risks and requires substantial exploratory development efforts. First, strategies will be developed to increase synthesis yields and to prevent lipid bilayer aggregation. Next, MP expression, purification and co-reconstitution of MPs in DLNs need to be established. Then, freezing conditions, additives and other sample preparation parameters need to be iteratively optimized to produce high quality grids for imaging. It is expected that the same or better resolutions can be achieved than with established bilayer mimetics, while providing new functionalities and unprecedented programmability. It is expected that DNA-lipid nanodiscs will initiate new research in structural biology, pharmacology, virology and bio-catalysis and therefore enhance the understanding of common diseases. The methods and models developed in this research will be made publicly available to benefit the larger scientific community researching the mechanisms of actions of drugs and vaccines.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.