Given the higher potency and unique CD spectrum observed for N-Myc73-94-N85C/G89C-mal when compared to the linear WT sequence, we further investigated the solution structure of the unbound peptide by NMR spectroscopy. Samples of N-Myc73-94 and N-Myc73-94-N85C/G89C-mal were prepared, and their spectra analyzed at 5 °C. The NMR spectra for N-Myc73-94 at this temperature showed well resolved 1 H resonances (Fig. ), whilst constrained N-Myc73-94-N85C/G89C-mal exhibited broadened resonances of lower intensity (Fig. -18 for full spectra at 5 °C). Although TOCSY spectra for both peptides had some similarity (Fig. ) many N-Myc73-94-N85C/G89C-mal 1 H signals were not observable (Cys res 85 and 89) precluding full assignment (see Fig. ). We hypothesize such behavior arises as a consequence of the maleimide constraint restricting the rate of interconversion between different conformers such that they match the NMR timescale, broadening the resonances to the point of coalescence. Such behavior has previously been observed in molten globules and partially folded proteins. To confirm this hypothesis, we carried out Reducing the temperature to 0 or -3 °C restored the exchange/broadening effect, supporting the notion that slow exchange between conformers occurs. (Fig. , note: in the absence of a reference value for a maleimide linked cysteine residue, the value for cysteine was used). Cα carbons experience a downfield shift when they are located in α-helical regions and it has been shown that Cα secondary shifts, ΔẟCα (i.e. the observed Cα shifts, ẟ -the expected Cα shifts for that residue in a random coil conformation, ẟRC), offer an accurate predictor of secondary structure. A continuous series of downfield (i.e. positive) Cα secondary shifts were observed for WT N-Myc73-94 between Ser76 N-Myc and Leu87 N-Myc at 5 °C, indicating propensity for helix-formation (Fig. , Table ). Of note, the magnitude of the observed downfield Cα secondary shifts (ΔẟCα ≤ 0.5-1.5 ppm) are lower than those typically seen in heavily stabilized α-helical structures (ΔẟCα ≥ 2.0-3.5 ppm), in agreement with the predominantly random coil conformation of N-Myc73-94 observed by CD (Fig. ). The downfield shifts are particularly pronounced at the N-terminus of the proposed α-helical region (Ser76 N-Myc to Gly89 N-Myc based on the crystal structure; For N-Myc73-94-N85C/G89C-mal at 25 °C, as for the wild type peptide at a 5 °C, the data are suggest there may be secondary structure propensity despite the increase in temperature.