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Template-assembled melittin: Structural and functional characterization of a designed, synthetic channel-forming protein
Michael Pawlak,
Ulrich Meseth,
Horst Vogel,
Boopathy Dhanapal,
Manfred Mutter,
Michael Pawlak
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Search for more papers by this authorUlrich Meseth
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Search for more papers by this authorCorresponding Author
Horst Vogel
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne (EPFL), Ecublens, CH-1015 Lausanne, SwitzerlandSearch for more papers by this authorBoopathy Dhanapal
Institute of Organic Chemistry, University of Lausanne, Lausanne, Switzerland
Search for more papers by this authorManfred Mutter
Institute of Organic Chemistry, University of Lausanne, Lausanne, Switzerland
Search for more papers by this authorMichael Pawlak,
Ulrich Meseth,
Horst Vogel,
Boopathy Dhanapal,
Manfred Mutter,
Michael Pawlak
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Search for more papers by this authorUlrich Meseth
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Search for more papers by this authorCorresponding Author
Horst Vogel
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne, Lausanne, Switzerland
Institute of Physical Chemistry IV, Swiss Federal Institute of Technology Lausanne (EPFL), Ecublens, CH-1015 Lausanne, SwitzerlandSearch for more papers by this authorBoopathy Dhanapal
Institute of Organic Chemistry, University of Lausanne, Lausanne, Switzerland
Search for more papers by this authorManfred Mutter
Institute of Organic Chemistry, University of Lausanne, Lausanne, Switzerland
Search for more papers by this authorAbstract
Template-assembled proteins (TASPs) comprising 4 peptide blocks, each of either the natural melittin sequence (melittin-TASP) or of a truncated melittin sequence (amino acids 6–26, melittin6–26-TASP), C-terminally linked to a (linear or cyclic) 10-amino acid template were synthesized and characterized, structurally by CD, by fluorescence spectroscopy, and by monolayer experiments, and functionally, by electrical conductance measurements on planar bilayers and release experiments on dye-loaded vesicles. Melittin-TASP and the truncated analogue preferentially adopt α-helical structures in methanol (56% and 52%, respectively) as in lipid membranes. Unlike in methanol, the melittin-TASP self-aggregates in water. On an air-water interface, the differently sized molecules can be self-assembled and compressed to a compact structure with a molecular area of around 600 Å2, compatible with a 4-helix bundle preferentially oriented perpendicular to the interface. The proteins reveal a strong affinity for lipid membranes. A partition coefficient of 1.5 × 109 M−1 was evaluated from changes of the Trp 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. These states are likely to be formed by aggregated TASP structures as inferred 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-assembled 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.
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