Reversible Photocontrol of DNA Binding by a Designed GCN4-bZIP Protein

Abstract

Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.

Structural specificity in coiled-coil interactions

“…They showed that adding the missing helix could constitute the trimer, and with it the enzyme [30]. Reversible photo-activation of coiled-coil transcription factor binding to DNA has also been demonstrated [31]. These applications illustrate the potential utility of coiled coils for biomedical or materials applications, which will be enhanced as we understand sequencestructure relationships better.…”

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

“…A variety of helical peptides and proteins bearing two Cys residues have been cross-linked, and their conformational and functional properties have been studied; these include the peptides JRK-7 and FK-11 and the proteins MyoD, engrailed and GCN4 27,[29][30][31] . Intermolecular cross-linking between proteins was not apparent in any of these cases and was likely suppressed by the fast intramolecular reaction between the chloroacetamide moiety of BSBCA and the second Cys residue.…”

Stimuli-responsive selection of target DNA sequences by synthetic bZIP peptides

“…Particularly relevant are those based on dimeric bZIP basic regions, in which the natural C/C-terminal leucine zipper is replaced by artificial dimerizers [14][15][16][17] . Some of these designs have gone even further and incorporate switchable elements that allow conditional off/on DNA binding by the application of external stimuli such as light or metal ions [18][19][20][21][22] . Despite advances in these and other switchable DNA binders 23,24 , designed systems capable of interacting with alternative DNA sites in an externally regulated manner are unknown.…”