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 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.
Institute of Biochemistry, National Yang Ming University, Taipei, Taiwan, ROC. chak4813@ms2.hinet.net
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Chak
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Hsieh
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Liao
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Kan
D001426:Bacterial Proteins / Q000302:isolation & purification; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D003087:Colicins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006361:Heating; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012997:Solvents; D013696:Temperature
D001426:Bacterial Proteins; D003087:Colicins; D012997:Solvents; C007104:colicin immunity proteins