WP2: Assembly of the lattice at the surface of membrane

Objectives: 
  1. Selection and conjugation of different phospholipid membrane anchoring moieties such as acyl chain, sterols, lipid-interacting polypeptide membrane anchors using chemical conjugation and biochemical attachment in vitro and in vivo.
  2. Self-assembly of the polypeptide scaffold on different bilayer types depending on the lipid composition.

Membrane attachment of the polypeptide building blocks designed in WP1 will be achieved by chemical coupling of hydrophobic functional groups such as the acyl, phospholipid chains, sterols, transmembrane or membrane-insertion polypeptide domains. Single or multiple functional groups will be introduced at different positions (preferably terminal or at flexible linkers). 

Chemical conjugation will be performed targeting the unique cysteine residues introduced into the polypeptide. Biological incorporation will be performed using ribosomal or enzymatic incorporation such as by the acyl-transferases either on the isolated proteins produced in WP1, in the in vitro transcription/translation reaction or in mammalian cells. Building blocks for the designed cytoskeleton will be incorporated onto the membranes of phospholipid bilayers of liposomes and on supported lipid bilayer coated mesoporous silica nanoparticles. 

Giant unilamellar vesicles will be used as model systems to study the formation of MAPS and to visualize the network formation by fluorescence/confocal imaging. Incorporation of selected bioactive molecules (targeting and internalization peptides, protein antigens, TLR agonists) to the surface of designed particles will be investigated. Different MAPS designs will be tested, differing in strength of interaction, geometry of the lattice and functional groups for membrane anchoring.