Interaction of a Histidine-Rich Antimicrobial Saliva Peptide with Model Cell Membranes: The Role of Histidines

Histatin 5 is a histidine-rich,intrinsically disordered, multifunctionalsaliva protein known to act as a first line of defense against oralcandidiasis caused by Candida albicans. An earlierstudy showed that, upon interaction with a common model bilayer, aprotein cushion spontaneously forms underneath the bilayer. Our hypothesisis that this effect is of electrostatic origin and that the observedbehavior is due to proton charge fluctuations of the histidines, promotingattractive electrostatic interactions between the positively chargedproteins and the anionic surfaces, with concomitant counterion release.Here we are investigating the role of the histidines in more detailby defining a library of variants of the peptide, where the formerhave been replaced by the pH-insensitive amino acid glutamine. Byusing experimental techniques such as circular dichroism, small angleX-ray scattering, quartz crystal microbalance with dissipation monitoring,and neutron reflectometry, it was determined that changing the numberof histidines in the peptide sequence did not affect the structureof the peptide dissolved in solution. However, it was shown to affectthe penetration depth of the peptide into the bilayer, where all variantsexcept the one with zero histidines were found below the bilayer.A decrease in the number of histidine from the original seven to zerodecreases the ability of the peptide to penetrate the bilayer, andthe peptide is then also found residing within the bilayer. We hypothesizethat this is due to the ability of the histidines to charge titrate,which charges up the peptide, and enables it to penetrate and translocatethrough the lipid bilayer.