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 structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent alpha-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed 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 Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of beta-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of alpha-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests alpha-helix may not be important for anticandidal activity of Hsn-5.
Department of Oral Biology, School of Dental Medicine, State University of New York at Buffalo 14214, USA.
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Balasubramanian
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Bobek
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry* / Q000494:pharmacology; D002175:Candida / Q000187:drug effects / Q000472:pathogenicity*; D003001:Cloning, Molecular; D054808:Histatins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011392:Proline / Q000032:analysis*; D017433:Protein Structure, Secondary; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014270:Trifluoroethanol
D000935:Antifungal Agents; C068075:HTN3 protein, human; D054808:Histatins; D010455:Peptides; D012471:Salivary Proteins and Peptides; C011083:polyproline; D014270:Trifluoroethanol; D011392:Proline