MODIFICATION OF SPECIFIC AMINO ACIDS INVOLVED IN THE FUNCTION OF COLICIN E1 AND ITS INTERACTION WITH IMMUNITY PROTEIN (MEMBRANE, CHANNEL, BACTERIA)
Abstract
The pH dependence of ionophoretic activity of colicins E1, A and the channel-forming domain of colicin E1 was titrated. The involvement in the acidic pH requirement of the only two histidyl residues, 427 and 440, in the channel-forming domain of colicin E1 and of glutamic acid 468 was investigated via chemical modification and site-directed mutagenesis, respectively. Chemical modification of His 427 and His 440 with DEP resulted in a 3-fold increase in activity. This activity change could not be explained by an increase in binding of modified peptide to asolectin vesicles, in anion selectivity, or in single channel conductance. The activity increase was also observed in planar membranes. The activity of the modified peptide was found to be independent of an imposed diffusion potential (-60 to +42 mV) in vesicles, where the potential-dependence is small for unmodified peptide, but not in planar membranes. The colicin E1 immune phenotype to the channel-forming domain of colicin E1 was demonstrated in osmotically shocked cells lacking the outer membrane receptor for colicin E1. There is no critical role for the two histidyl or single cysteinyl residues of the channel-forming domain in the immune response as determined by chemical modification. The immunity protein was localized to the inner membrane of the E. coli maxicell strain DR1984. The relative levels of colicin and immunity protein in maxicells was quantitated. The immunity protein is not a protease or protease co-factor as assayed by incubation of ('3)H -leucine-labeled colicin E1 with immune and non-immune inner membrane vesicles and by the in vivo proteolysis pattern of colicin E1 produced in imm('+) and imm('-) strains. Cloning the immunity protein structural genes behind the tac promoter with subsequent induction by IPTG, did not increase immunity protein levels, with and without its native operator-promoter.
Degree
Ph.D.
Subject Area
Biochemistry
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