CHANGE OF THERMAL-STABILITY OF COLICIN E7 TRIGGERED BY ACIDIC PH SUGGESTS THE EXISTENCE OF UNFOLDED INTERMEDIATE DURING THE MEMBRANE-TRANSLOCATION PHASE

Purified colicin E7 was analyzed by CD spectrum and gel filtration chr omatography 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. Alt hough the melting temperature of the protein shifted from 54.5 degrees C to 34 degrees C at low pH, the thermal denaturation curves of colic in E7 at different pH conditions still fit a two-state model. These ex perimental results imply that a minor structural change, triggered by acidic pH, for instance, may reduce the energy required for protein me lting. In contrast to the minor change in secondary structure at diffe rent pH conditions, we observed that, in vitro, all monomeric colicin E7s converted into multimer-like conformations after recovering from t he partial unfolding process. This multimeric form of colicin can only be dissociated by formamide and guanidine hydrochloride, indicating t hat this protein complex is indeed formed by aggregation of the monome ric colicins, Most interestingly, the aggregated colicins still perfor m in vivo bacteriocidal activity. We suggest that in a partial unfoldi ng state the colicin is prepared for binding to the specific targets f or translocation through the membrane, However, in the absence of spec ific targets in vitro these unfold intermediates may therefore aggrega te into the multimeric form of colicins. Proteins 32:17-25, 1998, (C) 1998 Wiley-Liss, Inc.