Role of alpha-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of alpha-helical st ructure of Hsn-5 for its candidacidal activity. The following three Hsn-5 v ariants, where one or more functionally nonessential residues were replaced with proline (potent alpha-helix breaker), were produced by Escherichia co li expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). T he activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in tri fluoroethanol (TFE) that is considered the helix-promoting solvent, and lys ophosphatidylglycerol (LPG) micelles, the environment that more closely res embles the biological membranes. Our results indicated that 3P variant disp layed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P). while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the alpha-helical content of Hs n-5 proline variants does not correlate with the candidacidal activity. Fur ther, the CD spectral analysis of peptides in LPG micelles indicated the fo rmation of beta-turn structures in 0P and 3P variants. In conclusion, 3P va riant which exhibited comparable candidacidal activity to 0P contains lower percentage of alpha-helical structure than 1P and 2P variants, which exhib ited lower candidacidal activity. This suggests alpha-helix may not be impo rtant for anticandidal activity of Hsn-5. (C) 2000 Elsevier Science B.V. Al l rights reserved.