Designing heterodimeric two‐stranded α‐helical coiled‐coils: the effect of chain length on protein folding, stability and specificity

Abstract

The E/K coil, a heterodimeric coiled-coil, has been designed as a universal peptide capture and delivery system for use in applications such as biosensors and affinity chromatography. In this design, heterodimer formation is specified through the placement of charged residues at the e and g positions of the heptad repeat. The affinity and stability of the E/K coil has been modified in order to allow a greater range of conditions for association and dissociation by varying the chain length to obtain three, four and five heptad coiled-coils (21, 28 and 35 residues per polypeptide chain). The effect of chain length on stability and folding was examined by circular dichroism spectroscopy, guanidine hydrochloride denaturation, and redox equilibrium experiments. It was found that increases in chain length produced increases in the stability of heterodimeric coiled-coils, but in a nonlinear fashion. The resulting disulfide-bridged heterostranded molecules and reduced heterodimers span a wide range of stabilities (deltaG=3.3-11.9 kcal/mol), greatly expanding their scope for use in protein engineering and biomedical applications.

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 chose the second model since sedimentation equilibrium experiments have shown that E5/K5 can form a tetramer with a dissociation constant (K d dimer-tetramer ) equal to 200 µM (22). Additionally, the observed molecular weight in sedimentation equilibrium experiments of E4/K4 was midway between that of the dimeric and tetrameric species, indicative of a dimertetramer equilibrium at high concentrations (23). An interaction between two EK dimers within the matrix of the biosensor is theoretically possible since the dextran chains have been shown to be flexible enough to allow for such an interaction (60).…”

“…The stability and affinity of this E/K system have been successfully modified by varying the R-helical propensity (serine vs alanine), the hydrophobicity of residues in the coiled-coil core (valine vs isoleucine), and the chain length (three to five heptads, or 21 to 35 residues) (23,24).…”

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

“…These rules have been validated by database studies, 10,14 and are used as the cornerstone of much coiled-coil design. 2,[15][16][17] In addition, charged residues at e and g, which flank the hydrophobic core, Figure 1C&D, often form salt bridges in natural systems. 2,16,18,19 This is also exploited in coiled-coil design: judicious placement of complementary charges-e.g., Lys-Glu pairs at e and g-can be used to pattern sequences to control homotypic or heterotypic assembly of peptides otherwise possessing the same core a & d residues.…”

Induced heterodimerization and purification of two target proteins by a synthetic coiled‐coil tag

“…From the small size of a coiledcoil tag, we hypothesize that steric hindrance effects within the heterodimer would also be minimized when fused to a weakly interacting protein pair. In this work, we have used a coiled-coil pair (E3/K3) 11,12,17 as a tag to heterodimerize the two GFP variants ECFP and Venus, purified the heterodimer by a two-step affinity chromatography to homogeneity, and analyzed the E3/K3 interaction. The E3/K3 pair has the advantages of being short (21 amino acids per tag) and behaving as a heterodimer when the free peptides are analyzed in solution.…”

“…the past; 2,9-15 for example, properties like the association constant, specificity, and oligomerization state of interacting peptides in solution could be controlled to some extent. 12,16 A synthetic, de novo designed coiled-coil offers the possibility of specifically copurifying weakly interacting proteins that otherwise would not withstand conventional purification procedures and to analyze their interaction in vitro, even at low concentration. From the small size of a coiledcoil tag, we hypothesize that steric hindrance effects within the heterodimer would also be minimized when fused to a weakly interacting protein pair.…”