On the structure, dynamics and mechanism of colicin E1 channel formation

Shaohua Xu, Purdue University

Abstract

A study of the topography of the colicin E1 channel by protease accessibility showed that both the NH$\sb2$- and COOH-termini are exposed toward the cis-side of the membrane bilayer. The 140 residue segment that is protected by the membrane from enzymatic digestion starts from residue ile-383 and extends to the COOH-terminus. When the $M\sb{\rm r}$ 18 kDa peptide was labeled in solution, the accessibility of tyrosine residues to the iodination reagent was 25 times lower at pH 6.0 than at pH 4.0, indicating an unfolding of the protein at latter pH. After the peptide bound to the membranes, the tyrosine residues became less accessible to the iodination reagent, indicating a large conformational change upon binding. Dynamic properties were illustrated by the channel peptide inserted at pH 4 being extruded from the membrane when the pH was shifted from 4 to 6. Reversible binding and insertion of the protein upon the pH 4 to 6 transition was shown by (1) increased protease accessibility, (2) a decrease in labeling by a phospholipid photoaffinity probe, and (3) intermembrane transfer of the extruded peptide upon an increase in ionic strength. A trans-membrane potential and acidic pH were necessary for channel formation in vitro. It was found that stimulation of channel activity in the presence of a K$\sp+$ diffusion potential was much greater in the presence of SO$\sb4\sp=$ as compared to NO$\sb3\sp-$ in the vesicle suspension medium. To localize the acidic pH effect, short peptides were generated by the combined techniques of protein chemistry and genetics. It was found that a $M\sb{\rm r}$ 7 kDa peptide (spanning residues 371-433) showed pH-dependent binding. This $M\sb{\rm r}$ 7 kDa peptide has the ability to initiate the membrane insertion process as indicated by photolabeling with different lipid probes. A site-directed mutagenesis study showed that lys-433 is not a critical residue in channel formation and channel conductance. An $M\sb{\rm r}$ 10 kDa peptide (spanning residues 434-522) had channel activity with liposomes and planar membrane bilayers. When the channel-forming peptide was incubated with SDS, it lost most of the acidic pH-dependence of membrane binding. The channel activity after preincubation in SDS, 2 $\times$ 10$\sp5$ Cl$\sp-$/channel/sec at pH 4.6-5.0, was the largest activity observed for the vesicle system. This pH optimum was higher than that in the absence of SDS. The peptide was unfolded at least partially by SDS as shown by increased protease accessibility. The binding of colicin E1 and its thermolytic peptide to membranes was found to be dependent on ionic strength. At high pH ($>$5.0) or ionic strength ($>$0.6 M for NaNO$\sb3$), the proteins did not bind to membranes. Higher channel activity was obtained with more negatively charged membrane vesicles.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biochemistry

COinS