Structure-based modelling of coiled-coil interactions was done as previously described, with modifications detailed in the Methods and Supplementary Information 26 (link). Using the technique of cluster expansion, structure-based models were converted to functions of sequence that included constant, single-residue and residue-pair terms. Training of the cluster expansion used 61,780 random bZIP-like sequences that were modelled structurally28 (link), 29 (link). A limited amino-acid alphabet was considered, which included the 10 residues most frequently found at each coiled-coil heptad position in native bZIPs. Constrained optimization employing integer linear programming (ILP) was used to design a , d , e and g sites. ILP optimization minimized the energy of design•target complexes, subject to constraints on the energy gap with respect to undesired complexes and the match of the design sequence to a position-specific scoring matrix derived from 432 native bZIP leucine zippers. Other positions in the coiled-coil repeat (b , c and f positions) were chosen to be consistent with the designed interface a , d , e and g residues, using a probabilistic framework. For each design target, the ILP optimization was repeated with increasing values of the specificity gap parameter Δ, in a procedure termed a specificity sweep. Sequences for experimental testing were selected manually from candidates generated using the specificity sweeps.
For experimental testing, His6-tagged peptides were expressed in RP3098 cells and purified by Ni-NTA followed by reverse-phase HPLC. Coiled-coil microarrays were printed, processed and probed as described previously4 (link). Fluorescence signals from the arrays were processed to remove background and normalized. Circular dichroism measurements were performed using standard techniques to measure spectra between 195 and 280 nm at 25 °C or thermal stability by monitoring ellipticityat 222 nm. Data were fit to appropriate thermodynamic equations to obtain apparent Tms. Detailed descriptions of all procedures are included in the Methods and theSupplementary Information .
For experimental testing, His6-tagged peptides were expressed in RP3098 cells and purified by Ni-NTA followed by reverse-phase HPLC. Coiled-coil microarrays were printed, processed and probed as described previously4 (link). Fluorescence signals from the arrays were processed to remove background and normalized. Circular dichroism measurements were performed using standard techniques to measure spectra between 195 and 280 nm at 25 °C or thermal stability by monitoring ellipticityat 222 nm. Data were fit to appropriate thermodynamic equations to obtain apparent Tms. Detailed descriptions of all procedures are included in the Methods and the
