Design of Light-Controlled Protein Conformations and Functions.
Remote control of cells is a desirable feature in synthetic biology. We established a light-switchable interfering peptide (iPEP) which controls gene expression by modulating the activity of a transcription factor. For photo-switching, the iPEP is cross-linked with a cis-trans isomerizable cross-linker in such a way that the light-activated cis form enables inhibitor folding rendering it active, whereas the dark-adapted trans form forces the inhibitor into an inactive form. Switching can be repeated in both directions. The iPEP acts as dominant-negative inhibitor targeting c-Jun and c-Fos of the transcription factor activator protein-1 (AP-1). Light-activated peptides exhibited much stronger inhibition of AP-1:DNA complexes and interference with gene transcription than their nonactivated counter parts. In this chapter, we provide protocols for cross-linking, peptide purification, observation of structural changes upon photo-switching, DNA binding analyses as well as gene expression studies in mammalian cells.