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Real-time tracking of single membrane translocases

Final Report Summary - SINGLEMEMB (Real-time tracking of single membrane translocases)

The threading of proteins through narrow channels within a cell is a crucial biological process, which is essential for cellular protein trafficking and protein degradation. The motor proteins driving this translocation process are called protein translocases. The Aubin-Tam group is developing biophysical tools to track the dynamics of protein translocation at the single-molecule level. The project objectives since the beginning of the Marie Curie CIG consisted of integrating a lipid bilayer electrophysiology within an optical tweezers system and of integrating a membrane translocase into the optical tweezers assay. During the second period of the Marie Curie CIG, free-standing phospholipid bilayers were interfaced with optical tweezers, with access to both lipid bilayer leaflets. FtsH has also been further structurally and biochemically characterized.

Since the beginning of the project, a strategy has been developed to functionalize styrene maleic acid lipid nanodiscs with small molecules. Labeling with a biotin molecule and with a fluorophore has been demonstrated. In parallel, a new type of microdevice has been designed and fabricated to form stable free-standing phospholipid bilayers. One major difficulty was to optically trap in close vicinity of the lipid membrane. A quantity of solvent, known as solvent annulus, is usually retained between the two leaflets at the edge of the membrane. Unfortunately, this annulus creates optical aberrations preventing optical trapping close to the membrane. To overcome this issue, the Aubin-Tam group identified a right combination of solvent and material for the flow cell, which led to successful optical trapping in close vicinity of the membrane.

The tools developed in this project could advance single-molecule investigations of membrane proteins, which represent 30% of all proteins and are prime drug targets. They will also find application in the study of the wide range of mechanical processes which occur at the cell membrane, such as lipid nanotube formation, flagellar locomotion, protein translocation or endocytosis.

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