Membrane Structure of the Colicin E1 Ion Channel

The colicins are a family of antimicrobial proteins that are secreted by Escherichia coli strains under environmental stress, due to nutrient depletion or overcrowding, and these proteins often target sensitive bacterial strains. The lethal action of colicins against their target cells are manifested in a number of different modes that include:

1. formation of depolarizing ion-channels in the cytoplasmic membrane,
2. inhibition of protein and peptidoglycan synthesis, and
3. degradation of cellular nucleic acids. In this context, the bacterial machinery responsible for colicin biological activity feature important mechanisms that are fundamental to various biological processes. These mechanisms include protein receptor binding, membrane translocation, membrane binding and protein unfolding, membrane-insertion, voltage-gated ion channel formation, catalysis, and inhibition of enzymes.

Colicin E1 is a member of the channel-forming subfamily of colicins and is secreted by E. coli that harbours the naturally occurring colE1 plasmid; the whole colicin consists of three functional segments, the translocation, receptor-binding, and channel-forming domains. Initially, the receptor-binding domain interacts with the vitamin B12 receptor of target cells. Following receptor recognition, the translocation domain associates with the tolA gene product, which permits the translocation of colicin E1 across the outer membrane and into the periplasm. In the periplasm, the channel domain undergoes a conformational change to an insertion-competent state, then inserts spontaneously into the cytoplasmic membrane of the host cell, forming an ion channel. The channel allows the passage of monovalent ions, resulting in the dissipation of the cationic gradients (H+, K+, Na+) of the target cell, causing depolarization of the cytoplasmic membrane. In an effort to compensate for the membrane depolarization effected by the colicin E1 channel, Na+/K+ ATPase activity is increased in the host cell, resulting in the consumption of ATP reserves, without concomitant replenishment. The final outcome is host cell death.

We are mapping the membrane-associated topology of the colicin E1 channel by cysteine-scanning mutagenesis (CSM). CSM is a powerful approach to the study of structure-function relationships in polytopic membrane proteins that has been pioneered by the Kaback group. We have previously employed depth-dependent quenching analysis, dynamic light scattering analysis as well as various steady-state and time-resolved fluorescence methodologies to probe the membrane structure of the colicin E1 channel. The Cys residues will be labelled with bimane, a thiol-specific fluorophore with interesting properties. The labelled channel domain will be tested for cytotoxicity and then will be incorporated into membrane vesicles according to our published system under conditions where channel activity can be induced. The membrane location of the bimane within each Cys mutant will be probed by employing membrane-permeant and impermeant fluorescence quenchers. Additionally, we will measure the following bimane fluorescence parameters: quantum yield, emission maximum, anisotropy, and decay times (lifetimes). We have conducted similar experiments using Trp as the fluorescence reporter and the correlation of all these parameters is a rich data source for membrane protein topology. In other experiments we will denature the bimane-labelled channel domain (BLCD) in 4 M urea and then label the Cys 505 with iodoacetamide-fluorescein, which will give us a nice FRET donor-acceptor pair. A subset of interesting BLCD proteins will be labelled with fluorescein and the distance estimated in the soluble and membrane-bound closed channel state. This will allow us to map each helix with reference to the Cys 505 (fluorescein) found in the hydrophobic anchor region of the channel domain.