Grafting Strategies of Oxidation-Prone Coiled-Coil Peptides for Protein Capture in Bioassays: Impact of Orientation and the Oxidation State

Many biomedical and biosensing applications require functionalizationof surfaces with proteins. To this end, the E/K coiled-coil peptideheterodimeric system has been shown to be advantageous. First, Kcoilpeptides are covalently grafted onto a given surface. Ecoil-taggedproteins can then be non-covalently captured via a specific interactionwith their Kcoil partners. Previously, oriented Kcoil grafting wasachieved via thiol coupling, using a unique Kcoil with a terminalcysteine residue. However, cysteine-terminated Kcoil peptides arehard to produce, purify, and oxidize during storage. Indeed, theytend to homodimerize and form disulfide bonds via oxidation of theirterminal thiol group, making it impossible to later graft them onthiol-reactive surfaces. Kcoil peptides also contain multiple freeamine groups, available for covalent coupling through carbodiimidechemistry. Grafting Kcoil peptides on surfaces via amine couplingwould thus guarantee their immobilization regardless of their terminalcysteine's oxidation state, at the expense of the control overtheir orientation. In this work, we compare Kcoil grafting strategiesfor the subsequent capture of Ecoil-tagged proteins, for applicationssuch as surface plasmon resonance (SPR) biosensing and cell cultureonto protein-decorated substrates. We compare the classicthiol coupling of cysteine-terminated Kcoil peptides to the aminecoupling of (i) monomeric Kcoil and (ii) dimeric Kcoil-Kcoillinked by a disulfide bond. We have observed that SPR biosensing performancesrelying on captured Ecoil-tagged proteins were similar for amine-coupleddimeric Kcoil-Kcoil and thiol-coupled Kcoil peptides, at theexpense of higher Ecoil-tagged protein consumption. For cell cultureapplications, Ecoil-tagged growth factors captured on amine-coupledmonomeric Kcoil signaled through cell receptors similarly to thosecaptured on thiol-coupled Kcoil peptides. Altogether, while orientedthiol coupling of cysteine-terminated Kcoil peptides remains the mostreliable and versatile platform for Ecoil-tagged protein capture,amine coupling of Kcoil peptides, either monomeric or dimerized througha cysteine bond, can offer a good alternative when the challengesand costs associated with the production of monomeric cysteine-taggedKcoil are too dissuasive for the application.

Automated summary

Functionalization of surfaces with proteins is needed in many biomedical and biosensing applications. The E/K coiled-coil peptide heterodimeric system has been found to be advantageous for this purpose. Various strategies of Kcoil grafting for capturing Ecoil-tagged proteins were compared, and the performances in biosensing and cell culture applications were evaluated. The results showed that both thiol coupling and amine coupling methods can be effective for protein capture, with each having its own advantages and limitations.