Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

47-POS Board 47 EXPLORING THE INTERACTION BETWEEN THE SIGNAL PEPTIDE FOR RETICULAR TRANSLOCATION AND THE SEC61 TRANSLOCON USING FORCE SPECTROSCOPY AT THE SINGLE MOLECULE LEVEL Luka Robeson 1 ; Nathalie Casanova-Morales 1 ; Francesca Burgos-Bravo 1 ; Christian A. M. Wilson 1 ; 1 Universidad de Chile, Biochemistry and Molecular Biology, Santiago, Chile The Sec61 translocon is a protein transport channel found in the membrane of the endoplasmic reticulum. It allows the translocation of proteins from the cytosol to the reticular lumen, a decisive step in the biosynthesis of most extracellular proteins. These secretory proteins possess a "signal peptide" at their N-terminus, which interacts with the translocon and begins translocation. Mutations in the signal peptide can impede translocation and cause diseases related to the intracellular accumulation of these proteins, indicating an essential role for this signal peptide-translocon interaction in the activation of translocation; however, the binding parameters that characterize these interactions remain to be defined. Single molecule force spectroscopy using optical tweezers was used to measure the force necessary to break the interaction between the signal peptide of Prepro-alpha-factor (PpαF) and Sec61. Dudko-Hummer-Szabo models were applied to the histograms of rupture forces obtained to calculate the mean lifetime of interaction in dependence of the force [τ(F)]. From the latter, the mean lifetime at zero force (τ 0 = 16.7 s) and the parameters of free energy (∆G‡ = 20K B T) and distance of the transition state (∆x‡ = 0.4 nm) of the dissociation process at zero force were obtained. Considering that the translocation rate is 1.1 amino acids/s, it is concluded that the interaction between the signal peptide and the translocon grants sufficient time to allow this process to happen. Next, we will compare these parameters with those from an Ala13Glu mutation in the hydrophobic region of the signal peptide of PpαF, which was reported to decrease the translocation rate up to 50 times, to study whether the loss of translocation efficiency may be related to a change in the dissociation parameters.

84