Fig. 1: Co-translocational unfolding mediated by a transmembrane pore.

a A disulfide-free mutant of thioredoxin with 3 mutations that increase its stability³¹ (A22P-I23V-P68A) was modified at a C-terminal cysteine with a 5′-maleimide-poly(dC)₄₀ to produce the construct Trx-V5-C109-poly(dC)₄₀. b A single α-hemolysin pore (α-HL) inserted in a lipid bilayer conducts an ionic current in response to an applied electrical potential and the negatively charged oligonucleotide is pulled into the pore. c At a constant potential of +120 mV, co-translocational unfolding occurs as manifested in the current trace, which reflects the molecular steps in the process: O, open pore; N, folded protein with the oligonucleotide threaded into the pore; I, an intermediate in the unfolding process with the C terminus threaded into the pore (the unfolded polypeptide segment is in red); T, translocation of the unfolded protein.