Conformational reprogramming of c-Myc via intracellular cyclisation.

Intrinsically disordered transcription factors represent a major challenge for chemical biology. Their lack of persistent structure limits both functional interrogation and ligand discovery. Here, we show that intracellular covalent cyclisation can conformationally reprogram the intrinsically disordered c-Myc bHLHZip region into a more ordered, DNA-binding state independent of its obligate partner Max. Using cell-penetrating bis-alkylating crosslinkers and rationally positioned cysteine pairs, we introduced helix-stabilising constraints into c-Myc during intracellular recombinant expression in E. coli. Among multiple designs, a single constraint positioned within helix two uniquely induced helicity and increased thermal stability, enabling sequence-specific E-box binding in the absence of Max. Notably, this gain of function does not recapitulate the canonical Myc-Max coiled coil architecture, revealing that non-native but functional conformational states can support sequence-specific DNA recognition. These findings demonstrate that intracellular chemical constraint can provide access to structured states that are normally inaccessible in the unbound protein and obscured within native protein complexes. By decoupling conformational ordering from partner occupancy, this approach establishes conformational reprogramming as a strategy to render otherwise intractable proteins amenable to functional interrogation and ligand discovery.