Change of thermal stability of colicin E7 triggered by acidic pH suggests the existence of unfolded intermediate during the membrane-translocation phase

Abstract

Purified colicin E7 was analyzed by CD spectrum and gel filtration chromatography in a mimicking membrane-translocation phase. It was found that the CD spectra of colicin E7 at pH 7 and pH 2.5 were similar. Although the melting temperature of the protein shifted from 54.5 degrees C to 34 degrees C at low pH, the thermal denaturation curves of colicin E7 at different pH conditions still fit a two-state model. These experimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein melting. In contrast to the minor change in secondary structure at different pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from the partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating that this protein complex is indeed formed by aggregation of the monomeric colicins. Most interestingly, the aggregated colicins still perform in vivo bacteriocidal activity. We suggest that in a partial unfolding state the colicin is prepared for binding to the specific targets for translocation through the membrane. However, in the absence of specific targets in vitro these unfold intermediates may therefore aggregate into the multimeric form of colicins.

Probing metal ion binding and conformational properties of the colicin E9 endonuclease by electrospray ionization time‐of‐flight mass spectrometry

“…Interestingly, apo‐E9 DNase at low pH had negative ellipticity at 222 nm, whereas the near‐UV CD signal and the Trp fluorescence were lost. Thus, at least under acidic conditions, apo‐E9 DNase seems to adopt features that are reminiscent of molten globule‐like states, that is, states that lack a fixed tertiary structure but in which secondary structure is retained (Le et al 1996; Chak et al 1998). In this regard, it is intriguing to speculate that the local acidic pH at the membrane surface triggers metal ion release and concomitantly renders the DNase in a molten globule‐like state competent for membrane translocation (Prats et al 1986; McLaughlin 1989; Bychkova et al 1996).…”

“…In this regard, it is intriguing to speculate that the local acidic pH at the membrane surface triggers metal ion release and concomitantly renders the DNase in a molten globule‐like state competent for membrane translocation (Prats et al 1986; McLaughlin 1989; Bychkova et al 1996). Interestingly, Chak et al have suggested that colicin E7 uptake into cells is aided by the acidic pH at the membrane surface and involves an unfolded intermediate (Chak et al 1998). Furthermore, it is reported that insertion of the pore‐forming domain of colicin A in the cytoplasmic membrane is preceded by a native to molten globule transition at acidic pH (Van der Goot et al 1991).…”

The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis

“…The expression vector pQE-30 (Qiagen, Germany) containing a six-histidine affinity tag at the N-terminus of the cloning site was used to overexpress nuclease-ColE7/Im7 complex. The detailed procedure for the construction and protein purification of the complex was previously described (19,31). The nuclease-ColE7 was further dissociated from Im7 by lowering the pH of the buffer to 3 to denature the purified complex and the two proteins were applied to a SP-Sepharose column (Amersham, USA) and eluted with buffers of pH 3 and 7, respectively.…”

Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

“…Alternatively, the positive cooperativity could be due to protein-protein interactions between membrane-bound nucleases. In this context, acidic pH-induced partial unfolding and subsequent multimerisation of the DNase E7 in solution have been demonstrated [39]. The DNase E8 is the only DNase whose binding profile is best approximated by a hyperbolic (non-cooperative) model.…”