Title
The structure analysis of Humanin analog, AGA-(C8R)HNG17, by circular dichroism and sedimentation equilibrium: Comparison with the parent molecule
Author
Fumio Arisaka, Takako Niikura, Tsutomu Arakawa, Yoshiko Kita
Year
2008
Journal
International Journal of Biological Macromolecules
Abstract
A 24-amino acid peptide, Humanin (HN), is a novel peptide that protects neuronal cells in vitro and in vivo from Alzheimer's disease-related toxicities. We have shown before that the structures of HN and a 1000-fold more active analog, HNG, with a Ser14Gly mutation are largely disordered. During additional mutational analysis, a shorter 17-amino acid form, AGA-(C8R)HNG17, was accidentally discovered to have a 100-fold higher activity than HNG. Here we have characterized the structural properties of the AGA-(C8R)HNG17 analog by circular dichroism (CD) and sedimentation equilibrium analysis. First, the structure in water was characterized, since these peptides have been dissolved in water prior to biological analysis. The AGA-(C8R)HNG17 peptide exhibited extensive β-sheet structure in water, completely different from the aqueous HN and HNG structures. The β-sheet structure was converted to a disordered structure upon dilution into phosphate-buffered saline (PBS) at low peptide concentration (e.g., below 0.2 mg/ml), which was similar to the structure of HN and HNG, observed under similar conditions. Sedimentation equilibrium analysis showed that the AGA-(C8R)HNG17 analog was essentially monomeric in PBS, while HNG showed extensive aggregation. Such aggregation of HNG was observed when the peptide was added to the serum-containing cell culture media. Thus, the mutations introduced into the AGA-(C8R)HNG17 analog generated a peptide different from the parent HNG and HN peptides in the self-association properties and hence the solubility, which most likely contributed to the increased biological activity of the AGA-(C8R)HNG17 analog.
Instrument
J-715
Keywords
Circular dichroism, Protein folding, Secondary structure, Biochemistry