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 coiled-coil 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 coil (KVSALKE heptad sequence) and E coil (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 coil or K coil, 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 coil interacted with the K5 coil according to a simple binding model. All the other interactions involving shorter coils were better described by a more complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. The affinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to 30 microM (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 coiled-coils. 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 coiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerable flexibility for protein engineering and biotechnological applications.
De Crescenzo
Gregory
G
Cell Surface Receptor Group, Health Sector, Biotechnology Research Institute, National Research Council (Canada), 6100, Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2.
Litowski
Jennifer R
JR
Hodges
Robert S
RS
O'Connor-McCourt
Maureen D
MD
D000595:Amino Acid Sequence; D003198:Computer Simulation; D019281:Dimerization; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D055672:Static Electricity; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance / Q000379:methods; D013816:Thermodynamics
D010455:Peptides