WP1: Design of the polypeptide scaffold technology

Objectives: 
  1. Design of the two-dimensional periodic lattice based on concatenated pairwise interacting segments (coiled-coil dimers) and single stable helices SAH that self- assembles into the selected geometric lattice.
  2. Selection, design and testing of polypeptide building modules based on natural and designed sequences and building of the accurate molecular model.
  3. Production and analysis of at least 6 different polypeptide designs in E. coli, based on several iterative design-build-test cycles interacting with WPs 2-4.

Periodical interacting networks will be designed by the application of graph theory establishing different geometries and topologies, in order to establish the construction rules and analyse the designs based on the concatenated interacting segments. Hexagonal as well as the tetragonal lattice will be constructed that will define the tightly knit lattice from the smallest number of different chains. Peptide modules for the interacting segments will be selected from our library of orthogonal parallel and antiparallel coiled-coil modules based on the orthogonality predicted by the algorithm based on 500 experimentally determined interactions, flexible linker segments (4-8 residues) and SAH. 

This library and design will provide a large selection of tunable interactions strength (3 orders of magnitude), chemical and geometric features for the polypeptide lattice. Molecular model of the periodic network will be built for the selected designs using Rosetta and Modeller algorithms (P1). Synthetic genes for the polypeptide coding segments will be designed for the production of recombinant proteins in E. coli and using an in house coupled in vitro transcription/translation (IVTT) system. Purified proteins will be characterized by biochemical, biophysical and high-resolution methods. 

Based on the results of the structural characterization of the assembled scaffolds (WP2, WP3), the next generation of the polypeptide scaffold will be iteratively designed (P1). The polypeptide scaffold will be genetically fused to the selected functional moieties, such as the enzymatic domains of the biosynthetic pathway (e.g. for the biosynthesis of resveratrol), metal nanoparticle binding peptide domains identified by phage display, fluorescent domains (pairs for the FRET and/or BRET detection) for the generation of the selected functionalities and demonstration of the application in WP4.