STRUCTURE-FUNCTION STUDIES ON COLICIN E1: A CLONED, VOLTAGE-DEPENDENT CHANNEL (GATING KINETICS, PROTEIN TRANSLOCATION, ION SELECTIVITY, PLANAR MEMBRANES, ENZYMATIC DIGESTION)
RAYMOND, LYNN ALISON
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Colicin E1, a 56 kD protein, forms voltage-dependent ion channels in planar membranes. Since its structural gene has been cloned and sequenced, it is a subject for studies of channel structure and function on a molecular level. In order to build a model of the E1 channel, two basic pieces of information are necessary: (1) the minimum amount of protein needed to make the channel, and (2) the diameter of the channel lumen. Previous work has shown that C-terminal fragments of colicin E1, of MW 16 to 20 kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. In this thesis, I place a lower limit of 8 (ANGSTROM) on the lumen diameter. Both pore size and ion selectivity of the channel formed by E1 and its C-terminal fragments were investigated. A minimum pore size was determined by measuring reversal potentials for gradients formed by salts of large, monovalent cations and anions; the pore was found to be permeable to ions as large as glucosamine('+), glucuronate('-), and NAD('-). The sensitivity of this method for measuring channel permeability depends on the pH of the test salt solution, since the channel was found to be permeant to both cations and anions, but its relative permeability to them is a function of pH. Also, under conditions to be described in this thesis, the gating kinetics and ion selectivity of channels formed by the different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments, and has little effect on the shortest C-terminal fragment. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. I propose a model for gating of the E1 channel, involving membrane translocation of large protein segments which are not themselves necessary for channel formation but apparently affect gating kinetics and ion selectivity. This study represents a first step toward implicating specific segments of the protein in particular channel functions.