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
详细信息 查看全文
- 作者：Gregory De Crescenzo ; Jennifer R. Litowski ; Robert S. Hodges ; and Maureen D. O'Connor-McCourt
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：February 18, 2003
- 年：2003
- 卷：42
- 期：6
- 页码：1754 - 1763
- 全文大小：186K
- 年卷期：v.42,no.6(February 18, 2003)
- ISSN：1520-4995
文摘
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 inbiosensors, 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 positionse and g of the heptad repeat. In this study, for each E coil or K coil, three peptides were synthesized withlengths varying from three to five heptads. The effect of the chain length of each partner upon the kineticand thermodynamic constants of interaction were determined using a surface plasmon resonance-basedbiosensor. Global fitting of the interactions revealed that the E5 coil interacted with the K5 coil accordingto a simple binding model. All the other interactions involving shorter coils were better described by amore complex kinetic model involving a rate-limiting reorganization of the coiled-coil structure. Theaffinities of these de novo designed coiled-coil interactions were found to range from 60 pM (E5/K5) to30 
M (E3/K3). From these Kd values, we were able to determine the free energy contribution of eachheptad, depending on its relative position within the coiled-coils. We found that the free energy contributionof a heptad occupying a central position was 3-fold higher than that of a heptad at either end of thecoiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerableflexibility for protein engineering and biotechnological applications.
M (E3/K3). From these Kd values, we were able to determine the free energy contribution of eachheptad, depending on its relative position within the coiled-coils. We found that the free energy contributionof a heptad occupying a central position was 3-fold higher than that of a heptad at either end of thecoiled-coil. The wide range of stabilities and affinities for the E/K coil system provides considerableflexibility for protein engineering and biotechnological applications.