The effects of introducing a chemical constraint within the helical region of N-Myc61-89 were next explored. To identify suitable positions for introduction of a constraint, we adopted a systematic approach. In this approach, cysteine pairs were introduced in the helical region of N-Myc61-89 at all non-hot-spot residues at i and i + 4 spacings, to give a series of 7 variants (see ESI, Table ). Each variant was then constrained using dibromomaleimide to afford the maleimide (mal) constrained variants (Fig. ), or allowed to oxidize to yield the disulfide bridged variant (ox), or reduced to its free-thiol form (red). The sequences of the resultant 21 variants along with their measured IC50, MRE222 values and % helicity estimates (determined using circular dichroism spectroscopy) are given in supporting information (Table and Fig.