Free-energy landscapes of membrane co-translocational protein unfolding

Protein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kBT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.D.R.L. work was supported by grants BIO2017-88946-R and BFU2016-81754-ERC from MINECO (FEDER funds), H.B. by grants from the National Institutes of Health, Oxford Nanopore Technologies and a European Research Council Advanced Grant. C.B.R. was funded by the Danish National Research Foundation (grant number DNRF78) and Aarhus University, Faculty of Science and Technology. D.R.L. is the recipient of a Ramon y Cajal fellowship (RYC-2013-12799). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government.Peer reviewe