TEMPLATE-ASSEMBLED MELITTIN - STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF A DESIGNED, SYNTHETIC CHANNEL-FORMING PROTEIN

Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a trunca ted melittin sequence (amino acids 6-26, melittin(6-26)-TASP), C-termi nally linked to a (linear or cyclic) 10-amino acid template were synth esized and characterized, structurally by CD, by fluorescence spectros copy, and by monolayer experiments, and functionally, by electrical co nductance measurements on planar bilayers and release experiments on d ye-loaded vesicles. Melittin-TASP and the truncated analogue preferent ially adopt alpha-helical structures in methanol (56% and 52%, respect ively) as in lipid membranes. Unlike in methanol, the melittin-TASP se lf-aggregates in water. On an air-water interface, the differently siz ed molecules can be self-assembled and compressed to a compact structu re with a molecular area of around 600 Angstrom(2), compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partitio n coefficient of 1.5 x 10(9) M(-1) was evaluated from changes of the T rp fluorescence spectra of the TASP in water and in the lipid bilayer. In planar lipid bilayers, TASP molecules are able to form defined ion channels, exhibiting a small single-channel conductance of 7 pS (in 1 M NaCl). With increasing protein concentration in the lipid bilayer, additional, larger conductance states of up to 1 nS were observed. The se states are likely to be formed by aggregated TASP structures as inf erred from a strongly voltage-dependent channel activity on membranes of large area. In this respect, melittin-TASP reveals channel features of the native peptide, but with a considerably lower variation in the size of the channel states. Compared to the free peptide, template-as sembled melittin has a much higher membrane activity: it is about 100 times more effective in channel formation and 20 times more effective in releasing dye molecules from lipid vesicles. This demonstrates that the lytic properties are not solely related to channel formation.