Utilizing the Trp-cage miniprotein in the design of light-responsive proteins and stable antimicrobial peptides

The miniprotein grafting method uses a non-functional miniprotein as a scaffold that can be modified to exert a specific function. The miniprotein Trp-cage is characterized by a highly stable fold including an α-helix of two turns and a short polyproline II helix. Being non-functional and only 20 residues in length, the Trp-cage is an ideal scaffold to be modified to achieve a specific function. In this thesis, two approaches to Trp-cage grafting are presented.

The first approach aims to use the stable fold of the Trp-cage to stabilize the secondary structure of an α-helical antimicrobial peptide (αAMP). This thesis demonstrates how fusing the well-known αAMP KR-12 to a Trp-cage domain may stabilize the α-helical conformation in the AMP-derived section of the fusion product termed antimicrobial Trp-cage (AMTC). Trp-cage grafting provided active αAMPs with low hemolytic activity and increased resistance against enzymatic degradation.

The second approach to Trp-cage grafting achieved photocontrol of the Trp-cage fold, i.e. the ability to reversibly switch the Trp-cage between a folded and an unfolded state by irradiation with light of different wavelengths. To obtain the photoswitchable protein termed switch cage, the Trp-cage was cross-linked at the α-helix with a new, light-responsive diazocine derivative. The study showed that it is possible to control a protein’s tertiary structure by the isomerization of a photoswitchable cross-linker attached to an α-helix.


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