Transport of proteins across membranes is a fundamental process, achieved in every cell by the 'Sec' translocon. In prokaryotes, SecYEG associates with the motor ATPase SecA to carry out translocation for pre-protein secretion. Previously, we proposed a Brownian ratchet model for transport, whereby the free energy of ATP-turnover favours the directional diffusion of the polypeptide (Allen et al., 2016). Here, we show that ATP enhances this process by modulating secondary structure formation within the translocating protein. A combination of molecular simulation with hydrogendeuterium-exchange mass spectrometry and electron paramagnetic resonance spectroscopy reveal an asymmetry across the membrane: ATP-induced conformational changes in the cytosolic cavity promote unfolded pre-protein structure, while the exterior cavity favours its formation. This ability to exploit structure within a pre-protein is an unexplored area of protein transport, which may apply to other protein transporters, such as those of the endoplasmic reticulum and mitochondria.

Department of Chemistry Imperial College London London United Kingdom

Department of Chemistry King's College London London United Kingdom

School of Biochemistry University of Bristol Bristol United Kingdom

SUPA School of Physics and Astronomy and BSRC University of St Andrews Scotland United Kingdom

University of South Bohemia in Ceske Budejovice České Budějovice Czech Republic

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Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon