Human saliva contains proteins with broad antibacterial and antifungal activity that protect oral tissues from pathogenic microorganisms. Salivary histatins, a family of small, histidine-rich, cationic proteins, are secreted by major salivary glands in humans and higher primates. Histatin-5 (DSHAKRHHGYKRKFHEKHHSHRGY, Hst-5), the most potent member of this family, is active against Candida albicans at concentrations found in saliva. Understanding their mechanism of action is important when considering histatins as candidates for drug therapy or as templates for drug design. The fungicidal mechanism of Hst-5 is proposed to be a multistep process initially characterized by binding to a yeast cell envelope protein, followed by intracellular translocation and efflux of ions and ATP. Hst-5 binds Zn²⁺ and Cu²⁺, ions present in saliva. We synthesized Hst-5 and an alanogue carrying the paramagnetic amino acid TOAC at its N-terminus (TOAC⁰-Hst-5) with the aim of comparing the physicochemical, conformational and biological behavior of the native and labeled peptides. Making use of circular dichroism (CD), fluorescence and electron paramagnetic resonance (EPR), we examined the binding of Zn²⁺, Cu²⁺ and Mn²⁺ to the peptides, as well as their conformational properties in solution, as a function of pH and trifluoroethanol (TFE), and in the presence of micelles of lysophospatidylcholine (LPC), lysophosphatidylglycerol (LPG) and LPC:LPG (2:1). Fluorescence titrations yielded similar values of ion binding constants for both peptides. Furthermore, neither CD spectra of both peptides nor EPR spectra of TOAC⁰-Hst-5 revealed any conformational changes upon ion binding or in pH titrations. In the presence of TFE both peptides adopted a-helical conformation. Fluorescence and EPR spectra showed that interaction between the peptides and micelles increased in the order LPC<LPC:LPG<LPG, pointing to the contribution of electrostatic interactions to binding. This was further evinced by the increased binding at lower pH, when peptides carry a higher positive charge. Future studies will aim at verifying whether the introduction of TOAC will affect the peptide biological activity.
Supported by FAPESP, CAPES and CNPq.