Real-time monitoring of the interactions of two-stranded de novo designed coiled-coils:: Effect of chain length on the kinetic and thermodynamic constants of binding

We have de novo designed a heterodimeric colied-coli formed by two peptides as a capture/ delivery system that can be used in applications such as affinity tag purification, immobilization in biosensors, etc. The two strands are designated as K coli (KVSALKE heptad sequence) and E coli (EVSALEK heptad sequence), where positively charged or negatively charged residues occupy positions e and g of the heptad repeat. In this study, for each E coli or K coli, three peptides were synthesized with lengths varying from three to five heptads. The effect of the chain length of each partner upon the kinetic and thermodynamic constants of interaction were determined using a surface plasmon resonance-based biosensor. Global fitting of the interactions revealed that the E5 coli interacted with the K5 coli according to a simple binding model. All the other interactions involving shorter colis were better described by a more complex kinetic model involving a rate-limiting reorganization of the colied-coli structure. The affinities of these de novo designed colied-coli interactions were found to range from 60 pM (E5/K5) to 30 muM (E3/K3). From these K-d values, we were able to determine the free energy contribution of each heptad, depending on its relative position within the colied-colis. We found that the free energy contribution of a heptad occupying a central position was 3-fold higher than that of a heptad at either end of the colied-coli. The wide range of stabilities and affinities for the E/K coli system provides considerable flexibility for protein engineering and biotechnological applications.