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bioRxiv · 10.1101/2024.10.10.617615

Modulation of SpyCatcher Ligation Kinetics by SpyTag Thioamide Substitution

Fiore, K.·Francis, D.·Casimir, S.·Perez, R.·Petersson, E. J.
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Abstract

Thioamide substitutions have been shown to impart valuable properties on peptides for biophysical experiments as well as cell or in vivo studies, but a rational understanding of thioamide effects on protein structure and protein-protein interactions is lacking. To elucidate their effects in {beta}-sheet structures, we have used SpyCatcher003-SpyTag003 as a host-guest system to study individual thioamide incorporation at eight different positions in the SpyTag peptide. We have demonstrated that incorporating thioamides into SpyTag at specific positions can result in a [~]2-fold faster ligating complex, as well as >2000-fold slower ligating complex. Biophysical analysis and structural modeling provide a reasonable explanation for most of the thioamide effects, altering hydrogen bond networks as well as modulating an n[->]{pi}* interaction within the SpyTag peptide. Our findings have important implications for potential applications of thioamide SpyTag variants, where the thioamide could impart protease stability in cells while also controlling the rate of ligation to SpyCatcher. These SpyCatcher-SpyTag host-guest experiments will also help to build a database for predicting thioamide effects on protein structure and function.

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Fiore, K., Francis, D., Casimir, S., Perez, R., Petersson, E. J.. 2024-10-10. Modulation of SpyCatcher Ligation Kinetics by SpyTag Thioamide Substitution. https://doi.org/10.1101/2024.10.10.617615

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