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Colicin E1 forms a dimer after urea-induced unfolding.

ABSTRACT: Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.

SUBMITTER: Steer BA

PROVIDER: S-EPMC1220293 | biostudies-other | 1999 Jun

REPOSITORIES: biostudies-other

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Project description:Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. The Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.

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Colicin E1 forms a dimer after urea-induced unfolding.

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