BINDING OF COLICIN-A AND COLICIN-E1 TO PURIFIED TOLA DOMAINS

Colicins are divided into two groups according to the proteins require d for their import into sensitive bacteria. The Tol and TonB pathways are involved in import of group A and group B colicins respectively. B ecause previous analyses have shown that colicin El and colicin A (two group A colicins) interact in vitro with the C-terminal domain of Tol A (TolAIII) while colicin B (group B colicin) does not, attention was focused on these interactions with purified proteins. TolA has been de scribed as a three-domain protein with an N-terminal inner-membrane an chor and a long periplasmic region formed by two domains (TolAII and T olAIII). TolAIII, TolAII and TolAII-III soluble domains with an N-term inal hexa-histidine extension were purified. The interactions of colic ins with the purified TolA domains were analysed by overlay Western bl otting, which indicated that both N-terminal domains of colicins A and E1 interacted with TolAIII, while a gel shift procedure detected no i nteraction with colicin E1. The binding kinetic values of the N-termin al domains of colicins A and E1 to TolAIII were estimated by surface p lasmon resonance and were shown to be similar.