The channel forming colicin protein provides a system that allows the study of a membrane active functional protein. Colicin E1 is a 522-residue protein that acts as a bactericidal agent. It can be divided into three functional domains. The C-terminal domain is responsible for binding to the cytoplasmic membrane and forming ion channels. The 192 residue C-terminal fragment is water-soluble. The crystal structure of this domain has been solved and found to contain 10alpha-helices. A similar structure was found for the C-terminal domain of colicin A1. A description of the mechanism of action of colicin E1 requires a complete understanding of the structural changes that occur upon insertion into the membrane. Competing structural models have been proposed to account for the transition between the membrane bound state and the active channel state. Our solid-state NMR studies of the colicin E1 C-terminal indicate that the 'umbrella' model is correct, because resonances from two trans-membrane helices were present in the spectra. The colicin samples were prepared in lipid bilayers, in the absence of any membrane potential. The results, therefore, are characteristic of the membrane bound, closed-channel state of the protein. We plan to extend the structural studies of the membrane bound form of colicin E1. Resonances from many of the 190 residues in the protein are resolved in the two-dimensional PISEMA spectrum, especially in the two membrane spanning helices. The next step is to perform three-dimensional solid-state NMR correlation experiments, which have been very effective in generating spectra with good resolution among resonances from in-plane helices.
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