Numerous options were reviewed to attach the fluorescent binding protein to the chamber of the biosensor. We initially considered using a carbon chain as a linker arm with two disulfide groups at either end. While this would get the job done, it would be expensive to develop and could lead to environmental toxicity issues. It also wasn’t a protein produced by a cell, and while that shouldn’t matter in the R&D phase, if we want to scale up the project to mass manufacturing cells producing a synthetic linker arm may be a cheaper solution.
After consultation with our advisors and experts in the field of protein engineering we decided to use a coiled-coil technique with a cysteine on the C-terminal of each heterodimer. The E coil alpha helix is made of five-heptad repeats which work in conjunction with the K coil alpha helices five-heptad repeats. The E heptad and K heptad contain sequences of EVSALEK and KVSALKE, respectively, where the hydrophobic residues leucine and valine form a hydrophobic interface, stabilizing the coiled coil heterodimeric structure. This hydrophobic effect is further amplified by repetition of the heptad repeat and provides great stability. Glutamate and lysine at the outer positions of the coils form electrostatic interactions further stabilizing the heterodimer structure.
E/K coiled coil system. All protein residues are shown with the E and K coils in red and blue (B-D) , respectively, using Chimera software. E and K coils each consist of five-heptad repeats with a hydrophobic core stabilized by glutamates and lysine residues surround the interface. A. Coil’s in a cartoon preset with each coil's cysteine labelled and displayed in a ball-and-stick preset. Note the sulfur atom in yellow. B. Coiled coil system in a mesh preset to visualize volume in 3D space. C. Volume in 3D space further enhanced with a solid surface preset, further, highlighting the interwoven nature of the coiled coil. D. Standard cartoon preset with the coiled coil alpha-helices oriented vertically.
Coiled coil system bound to fluorescent binding proteins. Protein structures were obtained from the RCSB Protein Data Bank. All protein residues are shown in a cartoon preset with the E and K coils in red and blue, respectively, using Chimera software. E and K coils each consist of five-heptad repeats with a hydrophobic core stabilized by glutamates and lysine residues surround the interface. Parathyroid hormone receptor with a Cys 48 residue.
Addition of a cysteine to the C- termini of either alpha helix provides a method of protein immobilization. One coils cysteine may bind a surface such as gold on the inside of a biosensor, while another coils cysteine can bind a proteins cysteine residue. The alpha helices may then associate, and a protein can therefore be immobilized. This method of protein immobilization is cost effective and simple compared to existing biochemical techniques. Note that these coils are expressed separately from one another and the binding protein construct.
Chao H, Bautista DL, Litowski J, Irvin RT, Hodges RS. Use of a heterodimeric coiled-coil system for biosensor application and affinity purification. J Chromatogr B Biomed Sci App. 1998 Sep 11;715(1):307–29.