Membrane Biophysical Studies of the Colicin E1 Channel

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Colicins are toxic proteins produced by certain strains of E. coli during bacterial cell growth and they are often produced in response to a host of metabolic challenges, including DNA damage, anaerobiosis, catabolite repression and nutrient depletion. Colicin E1 (colE1) is a member of the channel-forming colicins, which also includes colicins A, B, Ia, Ib, N and K. The three-dimensional structures of colicins reveal the presence of three domains: receptor binding, translocation, and channel domains. The receptor-binding domain initiates toxin entry into target cells by binding with the vitamin B12 receptor on the bacterial outer membrane. Next, the translocation domain associates with the trimeric beta-barrel TolC outer membrane receptor, through which the colicin is transported into the periplasm of the target bacterium. In a series of steps, colE1 sequentially unfolds, binds and spontaneously inserts into the membrane to form a closed channel state. The channel opens in the presence of a trans-negative membrane potential and causes depolarization of the host cell cytoplasmic membrane through the escape of cellular ions such as Na+, K+, and H+. Objectives: The overall goals of our NSERC-funded research program are: (1) To determine the membrane-bound topology of the colE1 channel protein (closed channel state), (2) To develop new fluorescence methods for the elucidation of colE1 membrane protein (low-resolution) structure, including the FRET Triangulation Method (3) To reveal the molecular details of the effect of a membrane potential on the structure and function of the colE1 ion channel with application to membrane proteins, in general. Significance: The proposed studies will provide molecular insight into the channel states of colE1 along with other related channel-forming colicins (A, B, E1, Ia, Ib, N, etc). Since NMR and X-ray studies have failed to produce high-resolution structures of the membrane-bound states, it is necessary to employ other approaches to provide "low-resolution" data, but important information for the understanding of colicin function. Furthermore, it is likely that we will produce a general method for the study of membrane potential on the structure and function of both integral and peripheral membrane proteins.