title: Protein co-translocational unfolding depends on the direction of pulling.
abstract: Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: David
authors.initials: D
authors.lastname: Rodriguez-Larrea
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
mesh_terms: D001427:Bacterial Toxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D058608:Nanopores; D017510:Protein Folding; D021381:Protein Transport; D058767:Protein Unfolding; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C491203:TXN protein, human; C021091:staphylococcal alpha-toxin; D013879:Thioredoxins