De Novo Designed Heterochiral Blue Fluorescent Protein

Diversification of chain stereochemistry offers a tremendous increase in protein design space. We have designed a minimal fluorescent protein, pregnant with β-(1-azulenyl)-l-alanine in the hydrophobic core of a heterotactic protein scaffold, employing automated design tools such as automated repetitive simulated annealing molecular dynamics and IDeAS. The de novo designed heterochiral protein can be selectively excited at 342 nm, quite distant from the intrinsic fluorophore, and emits in the blue region. The structure and stability of the designed proteins were evaluated by established spectroscopic and calorimetric methods.