Histatin 5 is an intrinsically disordered, multifunctional, cationic, saliva protein known to act as the first line of defense against oral candidiasis caused by Candida albicans and belongs to the family of antimicrobial peptides. The mechanism at the base of its antifungal activity is still partially unknown. For this reason, understanding the interaction between Histatin 5 and model membranes is of great importance for healthcare development. Solid-supported membranes have been demonstrated to be valuable systems used to mimic cell surfaces. The combined use of neutron reflectometry and quartz crustal microbalance techniques provided two-fold result: First, the electrostatic interactions between the positively charged proteins and anionic surfaces was identified as a driving force for the protein-lipid intermolecular interaction and second, under some particular conditions, Histatin 5 can lead to the formation of protein cushion underneath the model membrane, see Figure 1. This is important since, the supporting solid surface induces undesirable artefacts on supported lipid bilayers, e.g., not reproducing the deformability, nor the natural curvature of living cells, which indeed are properties that critically affect cellular adhesion processes, dynamics, and localization of transmembrane proteins. These results provide a "green" approach of forming cushioned bilayers and novel insights about protein-cell interactions.
Figure 1: The obtained scattering length density (SLD) from neutron reflectometry with a schematic sketch behind to show the system at each distance from the solid surface to highlight the meaning of the SLD at each region.
Several experiments have been performed at international large-scale facilities to characterize the protein-membrane interaction in several conditions. The experiments conducted by an international team lead by Marie Skepö (LUND and LINXS) were performed at the neutron reflectometers D17 (ILL), Super ADAM (ILL) and INTER (ISIS). Optimal conditions, in terms of bilayer composition, ionic strength of the solution, type of counter-ions, were pre-screened by QCM-D measurements performed at the Partnership for Soft Condensed Matter at the ILL and the selected samples prepared at the solid-liquid interface on the reflectometers. Measurements were performed in a classical solid-liquid geometry, upon application of contrast variation method and the data co-refined using the Aurore software.
The work was done in a collaboration between researchers at Lund University, Institut Laue–Langevin, and ISIS Neutron and Muon Source. The project was facilitated through funding by NordForsk Neutron Science Programme, the Crafoord Foundation, the Science Faculty project grant program for research with neutrons and synchrotron light (Lund University Strategic funds for MAX-IV and European Spallation Source), the Swedish research council for beamtime at Super ADAM, as well as O.E. and Edla Johansson Scientific Foundation. We acknowledge the Institut Laue-Langevin for the awarded beamtime (10.5291/ILL-DATA.9-13-656, 10.5291/ILL-DATA. CRG-2644) and for providing access to the PSCM laboratories and the Science and Technology Facilities Council for the awarded neutron beamtime at ISIS (10.5286/ ISIS.E.RB1800006).
Yuri Gerelli, Amanda Eriksson Skog, Stéphanie Jephthah, Rebecca J. L. Welbourn, Alexey Klechikov, Marie Skepö
Langmuir April 2020 (published online)
What is next?
The underlying mechanism of the cushion formation is attributed to have an electrostatic origin, and it is hypothesized that the observed behavior is due to proton charge fluctuations promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. The question we now face is whether we can validate our hypothesis: that the electrostatic mechanism, charge regulation is of importance for histidine-rich antimicrobial peptides. Work is in progress and a combination of computer simulations, QCM-D, and neutron reflectivity will be applied.