Role of α-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 α-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 α-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 α-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 β-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of α-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests α-helix may not be important for anticandidal activity of Hsn-5.     

Chemical cleavage of fusion proteins for high-level production of transmembrane peptides and protein domains containing conserved methionines

Due to their high hydrophobicity, it is a challenge to obtain high yields of transmembrane peptides for structural and functional characterization. In the present work, a robust method is developed for the expression, purification and reconstitution of transmembrane peptides, especially for those containing conserved methionines. By using a truncated and mutated glutathione-S-transferase construct as the carrier protein and hydroxylamine (which specifically cleaves the peptide bond between Asn and Gly) as the cleavage reagent, 10 mg of the first transmembrane helix of CorA, a Mg²⁺ transporter from Mycobacterium tuberculosis, can be conveniently obtained with high purity from 1 L of M9 minimal media under optimized conditions. The biophysical properties of the peptide were studied by circular dichroism and nuclear magnetic resonance spectroscopy, and the results show that this CorA peptide is well folded in detergent micelles and the secondary structure is very similar to that in recent crystal structures. In addition, this CorA construct is oligomeric in perfluoro-octanoic acid micelles. The compatibility with the transmembrane peptides containing conserved methionines, the high yield and the simple process make the present method competitive with other commonly used methods to produce such peptides for structural and functional studies.