Inactive C8A‑humanin analog is as stable as a potent S14G‑humanin analog

: We have previously shown that the structural stability of humanin (HN), a neuroprotective peptide ligand, is one of the attributes to the observed activity differences between HN analogs. It has been observed that the activity increased consecutively in the S7A‑HN analog, the parent HN and the S14G‑HN analog, consistent with the increased stability observed in that order. In the present study, the structure and stability of another inactive analog, C8A‑HN, was measured, which has been revealed to have no neuroprotective activity similar to that of the S7A‑HN analog and hence may have compromised stability. While all these analogs of HN demonstrated a similar disordered secondary structure in phosphate-buffered saline at 5˚C, as determined by circular dichroism spectroscopy, they revealed different structures at 37˚C. At 37˚C, less active HN and inactive S7A‑HN revealed a structure with a valley at ~217 nm, indicating a conversion from the disordered structure to a β‑sheet. Such a conversion was largely irreversible. By contrast, C8A‑HN and S14G‑HN demonstrated a similar structure at 37˚C and at 5˚C and remained largely disordered. The observed small structural changes of the C8A‑HN analog at 37˚C and its reversibility upon cooling do not support a hypothesis that the instability at 37˚C may have caused the reduced activity of this analog. Therefore an alternative explanation for its activity loss is required.