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Integral membrane proteins resolution of stoichiometry and structure

Final Report Summary - IMPRESS (Integral membrane proteins resolution of stoichiometry and structure)

At the start of this project we had mastered the ability to transmit membrane proteins into the gas phase of the mass spectrometer using a very limited range of detergents. We have now increased the number of detergents and mixed micelles compatible with mass spectrometry to >10 and have proposed mechanistic details of the different properties associated with these detergents.
During the course of the project we discovered that we could manipulate the natural membrane lipid environment through detergent dialysis protocols. In these experiments we selectively removed weakly associated lipids leaving only those required for structure and function. This procedure has been widely adopted and is yielding new insights into the annular lipid belts that surround membrane proteins and dictate their structure and function. Building on this theme we also demonstrated that we could use membrane mimetics to transfer membrane protein complexes from lipidic environments into the gas phase of the mass spectrometer.
Having established native lipid binding we were also keen to exploit its effects. To do this we devised a new approach for ranking the effects of ligand binding and to monitor their impacts on changes in structure. We devised a means of identifying the most critical ligands using a gas phase unfolding strategy whereby ligands are ranked according to their ability to resist unfolding in the gas phase. We developed software (PULSAR) freely available to the academic community and via a licensing agreement with pharma and biotech, to identify critical lipids involved in stabilizing membrane proteins for structure and function.
We also pursued laser activation of micelles to remove detergent and although success was achieved, no real advantages were observed beyond those of state-of-the-art collisional activation. Not proposed initially for IMPRESS, but an undoubted bonus of this research, have been our most recent developments. These developments have included the installation of a high-resolution mass spectrometry method capable of separating lipids, ligands metal ions and phospho forms. Together these developments establish a novel platform for drug discovery and elevate membrane protein mass spectrometry to a new level.