THE ROLE OF ELECTROSTATIC CHARGE IN THE MEMBRANE INSERTION OF COLICIN-A - CALCULATION AND MUTATION

The bacterial toxin colicin A binds spontaneously to the surfaces of n egatively charged membranes. The surface-bound toxin must subsequently , however, become an acidic 'molten globule' before it can fully inser t into the lipid bilayer. Clearly, electrostatic interactions must pla y a significant role in both events. The electrostatic field around th e toxin in solution was calculated using the finite-difference Poisson -Boltzmann method of the Delphi programme and the known X-ray structur e. A large positively charged surface was identified which could be in volved in the binding of colicin to negatively charged membranes. The applicability of the result was tested by also calculating the fields around modelled structures of the closely related colicins B and N. Su rprisingly, colicin N showed a similar charge distribution in spite of its isoelectric point of pi 10.20 (colicin A has pI 5.44). One reason for this is the strong conservation of certain negative charges in al l colicins. There is a single highly conserved aspartate residue (Asp7 8) on the positively charged face which provides a small but discrete region of negative charge. This residue, Asp78, was replaced by aspara gine in the mutant D78N. D78N binds faster to negatively charged vesic les but inserts only half as fast as the wild-type protein into the me mbrane core. This indicates that, first, the initial membrane binding has a significant electrostatic component and, second, that the isolat ed charge on Asp78 plays a role in the formation of the insertion inte rmediate.