DEPENDENCE OF THE LYTIC ACTIVITY OF THE N-TERMINAL DOMAIN OF HUMAN PERFORIN ON MEMBRANE LIPID-COMPOSITION - IMPLICATIONS FOR T-CELL SELF-PRESERVATION

The kinetics and thermodynamics of pore formation by the 22-residue N- terminal domain of human perforin-(1-22)-peptide have been studied for a variety of model phospholipid membranes. Peptide binding and aggreg ation, and cell lysis were monitored through the change in the fluores cence of Trp, or vesicle-encapsulated carboxyfluorescein, respectively . Peptide binding was analyzed in terms of a model that incorporates n on-ideal interactions and aggregation in a membrane bilayer. The minim um number of peptide monomers required to form an active pore averaged from four to six, according to the lipid composition of the vesicle. This combined kinetic and thermodynamic approach has provided quantita tive information that allows a direct comparison of the binding behavi or of the perforin-(1-22)-peptide in different lipid vesicles and affo rds molecular insight on the factors controlling pore formation. Pore formation is most favorable in thinner membranes with low melting temp eratures. No significant difference in activity is observed for differ ent zwitterionic headgroups. Rather, the gel state of the lipid chain, which diminishes the incorporation and aggregation of the perforin-(1 -22)-peptide shows the strongest influence. This effect is observed in bath the thermodynamic (incorporation isotherm) and kinetic (carboxyf luorescein release) studies. Insertion and aggregation are more facile in membranes with less densely packed lipids. The dependence of pore- forming activity on lipid composition provides important clues to unde rstanding the self-protection mechanism employed by cytotoxic T lympho cytes (CTL) against perforin-mediated lysis.