Secondary structure of the pore‐forming colicin A and its C‐terminal fragment

Abstract

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60,000), and a C-terminal bromelain fragment (Mr 20,000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly .alpha.-helical with an enrichment of the .alpha.-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible .beta.-sheet structure in the C-terminal domain is unstable and is easily transformed into .alpha.-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favored a model in which the pore is built of a minimal number of six transmembrane .alpha.-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic moment calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.