The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.

The interaction of colicin-A thermolytic fragment with negatively charged liposomes was studied by fluorescence spectroscopy. 1,2-Dioleoyl-sn-glycero-3-phospho-l-sn-glycerol (Ole,GroPGro) containing liposomes do not significantly alter the fluorescence properties of the protein, and thus cannot give much information about this interaction. 1,2-Bis(9,10-dibromooleoyl-sn-glycero-3-phospho-1-sn-glycerol (Br,Ole,GroPGro) is easily synthesized by addition of bromine atoms to the double bond located at the mid-point of the fatty-acid acyl chain of Ole,GroPGro. The brominated phospholipid forms vesicles that strongly quench the protein fluorescence emission. The results presented here show that conversion of Ole,GroPGro to Br,Ole,GroPGro does not change either the affinity for the protein or the extent of lipid binding. This observation allows for the estimation of the distribution of the quenching phospholipid molecules around the fluorophores [Yeager, M. D. & Feigenson G. W. (1990) Biochemistry 29, 4380-43921. Binding of the protein to the vesicles is an irreversible process, since inserted molecules do not dissociate from the vesicle. From steadystate measurements, it can be concluded that in the membrane-bound form, the tryptophans are located within quenching distance of the bromine atoms, i.e. close to the lipid head-grouphydrocarbon boundary, completely accessible to the quencher, protected from the polar phase and that the maximum number of phospholipid molecules in contact with the fluorescent domain of the protein is nine.Colicin A is a plasmid-encoded protein produced by some strains of Escherichia coli, which is active against those strains of E. coli endowed with the necessary receptor. The mechanism of action of colicin A in vivo comprises three steps : binding to the receptor, translocation across the outer membrane (with the intervention of the tolQ, tolR, tolA and tolB proteins) and formation of a voltage-gated pore which depolarizes the cell and kills it. Each of these steps is performed by a different domain in the protein. The central domain binds to the receptor, the N-terminal domain is responsible for protein translocation and the C-terminal domain forms the pore (Pattus et al., 1990).This C-terminal domain can be obtained from the whole colicin-A molecule by thermolysin proteolysis, and thus it is called the thermolytic fragment of colicin A (Tucker et al., 1986). It has been crystallized and its structure determined by X-Ray crystallography. Based on the three-dimensional structure of this fragment, a model for the insertion has been proposed , in which the compact soluble structure, upon interaction with the phospholipid bilayer, un- folds in an umbrella-like fashion. This is the so-called 'umbrella model'.The study of the membrane-bound conformation of colicin A by spectroscopic techniques shows that there is no drastic conformational change upon membrane insertion : the secondary structure of the protein and its fluorescence parameters are largely conserved (Lakey et al., 1991a; Goormagtigh ...

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