Histatin 5 is a histidine-rich,
intrinsically disordered, multifunctional
saliva protein known to act as a first line of defense against oral
candidiasis caused byCandida albicans. An earlier
study showed that, upon interaction with a common model bilayer, a
protein cushion spontaneously forms underneath the bilayer. Our hypothesis
is that this effect is of electrostatic origin and that the observed
behavior is due to proton charge fluctuations of the histidines, promoting
attractive electrostatic interactions between the positively charged
proteins and the anionic surfaces, with concomitant counterion release.
Here we are investigating the role of the histidines in more detail
by defining a library of variants of the peptide, where the former
have been replaced by the pH-insensitive amino acid glutamine. By
using experimental techniques such as circular dichroism, small angle
X-ray scattering, quartz crystal microbalance with dissipation monitoring,
and neutron reflectometry, it was determined that changing the number
of histidines in the peptide sequence did not affect the structure
of the peptide dissolved in solution. However, it was shown to affect
the penetration depth of the peptide into the bilayer, where all variants
except the one with zero histidines were found below the bilayer.
A decrease in the number of histidine from the original seven to zero
decreases the ability of the peptide to penetrate the bilayer, and
the peptide is then also found residing within the bilayer. We hypothesize
that this is due to the ability of the histidines to charge titrate,
which charges up the peptide, and enables it to penetrate and translocate
through the lipid bilayer.