Abstract
A prolonged digestion of colicin E1 with dilute trypsin results in the generation of a trypsin-resistant peptide fragment of the colicin which is approximately one-third of the molecule. The amino acid composition of the fragment, Mr = 20,000, is more nonpolar in nature than the colicin, with the major change in composition being the reduction of the arginine content from 25 residues in the Mr = 56,000 colicin molecule to approximately 1 residue in the fragment. The NH2-terminal amino acid sequence of the tryptic fragment shows no similarity to that of colicin E1. Alignment of this sequence with the complete amino acid sequence of the colicin E1 molecule obtained by others, shows that the fragment occupies all, or almost all, of the COOH-terminal section of the molecule. The fragment behaved similarly to colicin E1, in being able to dissipate a potassium diffusion potential in unilamellar membrane vesicles made of dimyristoylphosphatidylcholine. The fragment was able to dissipate the diffusion potential above and below the temperature region (Tm = 23.5-24 degrees C) of the lipid phase transition in these vesicles, consistent with a channel-like function. The fragment did not show measurable binding to colicin receptor sites on the cell surface, but was much more efficient than colicin E1 in its ability to inhibit proline transport by inner membrane vesicles of Escherichia coli. These data imply that a membrane channel-forming function of the molecule is located in this 20,000 molecular weight region at the COOH-terminal end of the colicin molecule.
Highlights
A prolonged digestion of colicin E l with dilute trypsin results in thegeneration of a trypsin-resistantpeptide fragment of the colicinwhich is approximately one-third of the molecule
The fragment behaved to colicin El, in being able to dissipate a potassium diffusion potential in unilamellar membrane vesicles made of dimyristoylphosphatidylcholine.The fragment was able to dissipate the diffusion potential above and below the temperatureregion
Generation of a Trypsin-resistant ColicinPeptide-The digestion of the colicin E l molecule by dilute (1:320, w/w, 25 "C) trypsin is shown as a function of the time of incubation by the gel patterns of Fig. 1and the associated densitometry scan of Fig. 2
Summary
The fragment did not show measurable binding to colicin receptor sites on the cell surface, but was much more efficient than colicin E l in its ability to inhibit proline transport by inner membrane vesicles of Escherichia coli These data imply that a membrane channel-forming function of the molecule is located in this 20,000 molecular weight region at the COOH-terminal end of the colicin molecule. The domain structure concept has been examined to a limited extent with the group of colicins, E l , K, Ia, and A, which cause de-energization of the cell membrane [13, 14] These experiments concluded that proteolytic treatment can generate NH2-and COOH-terminal fragments which bind to receptor and interactwith the inner membrane, respectively, in analogy to the previously described proteolysis studies [5, 7,8,9]. The ability of the COOHterminal tryptic fragment of colicin E l to de-energize target membranes is directly demonstrated
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