The effect of replacing phenylalanine with alanine at residue 427 of PA₆₃ on the rate of translocation of LF_N. (A) The experiments were performed as described in Fig. 3. For the WT channel, as shown in Fig. 3 and reproduced here, the introduction of an SO₃⁻ at residue 59 (red) drastically reduced the rate of LF_N translocation from that of MTS-ACE–reacted LF_N (black), which was the same as that of WT LF_N (not depicted). Mutating the phenylalanine at residue 427 to alanine in the (PA₆₃)₇ channel dramatically increased the rate of translocation of LF_N with an attached SO₃⁻ (orange) and decreased the rate of translocation of MTS-ACE–reacted LF_N (gray), which was the same as that of WT LF_N (not depicted). (B) Cartoon of the (PA₆₃)₇ channel with LF_N being translocated through it. The constriction near the junction of the vestibule with the stem is the Φ-clamp, which is formed by seven F427s. We envision that for negatively charged residues (aspartates or glutamates) on LF_N to get past the Φ-clamp, they must be neutralized; that is, they pick up protons from the cis solution. This proton is subsequently discharged into the trans solution after the residue has traversed the channel. If a non-titratable SO₃⁻ is introduced into LF_N, it is retarded at the Φ-clamp. Mutation of F427 to alanine alters the constriction and now more readily allows a negatively charged group such as SO₃⁻ to get past it.