Knowledge of the structure of proteins can provide an in-depth understanding of biology and lead to critical insights into the origins of human disease. Numerous proteins important in biology and disease are, however, not amenable to structural analysis by traditional methods. A family of proteins that are notoriously difficult to study are membrane proteins. Membrane proteins are extremely important to study as they play pivotal roles in most biochemical processes of the cell, account for up to 25 percent of all proteins in humans, and represent nearly two-thirds of the proteins that can be targeted by drugs. These proteins are, however, difficult to access experimentally due to their hydrophobic nature and because they need to be associated with lipids from the cell membrane. Most techniques are not readily compatible with the combination of lipids and proteins, especially those that are used to reveal the three-dimensional structure of proteins (i.e. NMR spectroscopy or x-ray crystallography). Alternative techniques are needed to gain insight into the structure of membrane-bound proteins. We propose to meet this challenge by employing an alternative technology that use mass spectrometry to measure the hydrogen/deuterium exchange (HX) of proteins in solution. The objective of this project proposal is to apply HX-MS technology to study two ”difficult” membrane proteins of key biological function: (1) the T-cell receptor (TCR) and (2) the Epidermal Growth Factor Receptor (EGFR). By inserting these receptors into nanoscale lipid bilayer discs and measuring the HX of the proteins, we will extract detailed information about their higher-order structure and molecular interactions using only small amounts of material at dilute biological conditions. The proposed project is innovative as we combine nanotechnology with alternative analytical methodology to supply critical and currently missing structural details about two important cell-surface receptor proteins.
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