THE STRUCTURAL AND FUNCTIONAL PROPERTIES OF THE COLICIN E1 MOLECULE (DOMAINS, CIRCULAR DICHROISM, MEMBRANE PROTEIN)
Abstract
The domain arrangement of the colicin E1 molecule was studied by proteolytically cleaving the colcin as a means to obtain individual functional domains. The receptor-binding domain of colicin E1 was found to be situated in the central portion of the colicin molecule, with a 16-kDa fragment from this region showing the ability to block the killing actions of colicins E1 and E2 when added to sensitive E. coli cells. A previously reported 20-kDa COOH-terminal tryptic peptide of colicin E1 was used to study the structural and functional properties of the channel-forming region of the toxin. This channel-forming domain was found to undergo an increase in (alpha)-helical content in non-polar detergent solutions as the pH of the media was lowered below pH 4.5. The binding and insertion of colicin E1 and the 20-kDa peptide into artificial vesicles were shown to have a similar dependence on pH. A model of the colicin channel was proposed based on these data and the amino acid sequence of the toxin. This model, which is composed of five membrane-spanning amphipathic helices, provided an explanation for the observed pH-dependence of colicin E1 channel activity. Further characterization of the channel domain has provided data that is consistent with this model. In particular, treatment of channel-forming peptide with proteases following the insertion of the peptide into artificial vesicles yielded a resistant fragment of M(,r) = 14,000 with this size fragment agreeing with that predicted to be generated based on the five-helix model. It is concluded that colicin E1 channel binding and insertion require an acidic pH, and that channel insertion probably results in an increase in the helical content of this domain.
Degree
Ph.D.
Subject Area
Biology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.