Penetration preparations: Studies on the insertion competent state of the colicin E1 channel polypeptide and the interaction between colicin E1 and TolA

Sharon Lee Schendel, Purdue University

Abstract

Colicins are plasmid-encoded protein antibiotics produced by and active against Escherichia coli and related strains. Colicin E1 kills sensitive cells through the formation of an ion channel in the cell's cytoplasmic membrane that is of sufficient conductivity to dissipate the cellular membrane potential. How colicin E1 is able to make the transition from a soluble to a membrane-inserted protein is of interest in studies of protein insertion into membranes. In vitro, the C-terminal domain of colicin E1 takes on a more flexible, hydrophobic character upon exposure to acidic pH or exposure to low concentrations ($<$10$\sp{-3}$ mol%) of the detergents SDS or $\beta$-octyl glucoside. The nature of the intermediate state imposed by such treatment, the "insertion-competent" state, was examined by circular dichroism and fluorescence of intrinsic tryptophans and the hydrophobic fluorescence probe, ANS. These studies revealed that the secondary structure of the colicin E1 channel domain is preserved upon exposure to acidic pH or detergent and the tertiary structure is only mildly perturbed. These small structural changes correlate well with increased in vitro channel activity, highlighting the importance of this state for channel formation. The colicin E1 channel polypeptide serves as an example of the tertiary changes involved as a protein undergoes a large change in character, in this case, soluble to hydrophobic. These structural changes need not be large, in contrast to those seen in the well-documented "molten globule" state which has no native-like tertiary structure and is proposed to have an important role in membrane protein insertion. In vivo, colicin E1 requires the presence of several cellular proteins to aid its journey across the E. coli cell envelope. TolA is of greatest importance to colicin E1 activity. TolA has an unusual central domain whose sequence resembles that of a synthetic peptide. This region is highly $\alpha$-helical and contains thirteen pentapeptides of the sequence: K-A$\sb3$-D/E. The role of the TolA $\alpha$-helical domain in colicin E1 translocation was explored through the creation of TolA molecules having deletions in this central region. Colicin E1 is unique in its ability to locate TolA molecules lacking nearly the entire central domain, suggesting that its mode of cell entry differs markedly from the other TolA-dependent colicins. It is unclear how colicin E1 makes the transition to a membrane-inserted protein in vivo, but this transition is probably fostered by interactions between the colicin molecule and cellular proteins involved in its uptake. The ability of a soluble form of TolA to invoke structural changes in colicin E1 in vitro that resemble the insertion-competent state was explored.

Degree

Ph.D.

Advisors

Cramer, Purdue University.

Subject Area

Biophysics|Cellular biology

COinS