Bharat Kumar
Research Proposal
Title: Electrical interactions between antimicrobial peptides and supported lipid bilayer.
Origin of the proposal:
In this proposed research, we plan to study the electrical interactions between the
antimicrobial peptides and supported lipid bilayer membrane. Antimicrobial peptides (AMPs)
are small amphiphilic peptides which are widely present in plant and animal kingdom as
weapons against microbes[1]. In the last few years many studies have been carried out to
understand the mechanism of their action on cells. Usually, the AMPs are cationic and are
known to kill the microbial cells(a) by permeabilizing and disrupting the cell membrane[2].
The microbial cell membrane comprises of phospholipid molecules such that the outer layer
of the cell membrane is charged. In contrast to this, the cell membranes of plants and animals
are composed of phospholipid molecules such that the outer surface of the lipid membrane is
not charged. This difference between the microbes and multicellular organisms is exploited
by the plant and animal kingdom to produce AMPs which specifically act and destroy
microbes without harming the host cells. Based on different experimental studies at least four
models have been proposed to explain the action of AMPs on microbial cell membranes, viz.,
(a) toroidal model [3], (b) Carpet model [4], (c) Barrel-Stave model [5] and (d) aggregate
channel model [6]. The mechanism followed by a particular AMP to destroy the microbial
cell depends on its structure, charge, amphiphaticity (or amphiphilicity), hydrophobicity, etc
and also on the structure and properties of the target cell membrane.
Various studies have been carried out to understand the action of AMPs on the cell
membranes by considering the above effects. The first step in the process of AMPs’ action on
microbes is to attach itself to the target cell membrane. This step is mainly guided by the
electrostatic interactions between the charged cell membrane and the AMP. These electrical
interactions are influenced by the factors like the membrane potential, membrane
phospholipid composition, electric double layer forces, salt concentration in the surrounding
electrolytic medium etc. Despite the significance of electrostatic interactions between the
AMP molecules and lipid membrane, fewer studies have been carried out in this direction.
(a)
In addition to their action on cell membrane, AMPs are also reported to target intracellular
components like DNA, RNA etc.
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Bharat Kumar
We propose to study the electrical interactions at molecular-level between the AMP
molecules and supported lipid bilayer which mimic the cell membrane. The choice of
supported lipid bilayer over the native membrane is made to avoid the complexity involved in
biological systems. Also, the supported lipid bilayer provides a system, where the membrane
properties can be controlled and changed to get a broader picture of electrical interactions
between lipid membrane and AMP molecules.
Objectives:
In this proposed research we would like to study the electrical interactions between
the AMPs and the supported lipid bilayer (SLB). The work will be carried out to address the
following specific questions:
(a) How does the ion concentration, type of ions and pH of an electrolytic solution affect
the AMP molecules?
(b) How the SLB surface charge density and potential are affected by AMP molecules?
(c) How do the ions in the electrolytic medium together with bilayer surface charge affect
the interaction forces between the AMP molecules and the lipid bilayer?
Results of these investigations will give the deeper picture of AMP-lipid membrane
interactions and helps us to understand the mechanism of AMP action on the microbial cells
under physiological conditions. Quantitative analysis of these results may also help us to
correlate the membrane stability to the lipid composition and ionic effects in the presence of
AMP molecules. In addition, we would also like to compare our results to the studies carried
out on DNA-membrane interactions and try to build a common platform where in the
electrical interactions between a polyelectrolyte (both rigid and flexible) and charged liquidcrystalline surface can be addressed.
Methodology:
Molecular-level understanding of AMP-SLB membrane interactions in an electrolytic
media can be realized by employing scanning probe technique like atomic force microscopy
(AFM). Figure 1 shows the cartoon of the AFM setup having a cantilever fixed at one end
and with a sharp tip at the other end. The tip diameter is usually less than 30 nm. The forces
between the sample and tip results in the deflection of the cantilever which can be measured
by reflecting a laser beam on the back of the cantilever and monitoring the reflected beam
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Bharat Kumar
using a split photo-detector (Figure 1). If the force constant of the cantilever is known then
the deflection measurements can be used to calculate the force acting on the cantilever.
Split photodetector
Laser
Cantilever
AMP
Processor &
Controller
SLB
Tip
Substrate
Piezo scanner
Figure 1: Schematic of atomic force microscope (AFM).
Supported lipid bilayer (SLB) membranes have been widely used as model systems to
mimic the lipid cell membrane [8]. The SLB membranes will be prepared by following the
protocol described elsewhere [8,9] using different lipid molecules. Anionic lipids like 1Palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) and 1,2-Dipalmitoyl-anglycero-3-[phospho-rac-(1-glycerol)] (DPPG) will be used to mimic bacterial cell membranes
and zwitterionic lipids like 1,2-Dipalmitoyl-an-glycero-3-phosphocholine (DPPC) and 1Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) will be used to mimic mammalian
cells.
The effect of ion concentration and pH of the electrolytic solution on the AMP
molecules will be addressed by studying the immobilized AMP molecules (MSI-78 and
Magainin I) on solid substrates (silicon, gold, mica) using an AFM. Force-distance curves
and electrostatic force images obtained using AFM gives us information about the isoelectric
point (or point of zero charge) of AMP molecules and relative charge on the AMP as a
function of ion concentration and pH, respectively [10,11,12].
The effect of AMP molecules on the bilayer charge density and potential can be
understood by carrying out the studies on supported lipid bilayers with AMP molecules
adsorbed onto the bilayer surface from the surrounding liquid medium. The lipid bilayer
surface charge density and effective bilayer potential will be obtained as a function of
concentration of AMP molecules from the force-distance (F-d) curves using AFM. The
experimental F-d data will be analyzed by two methods:
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Bharat Kumar
(a) Using the analytical expression for force between an AFM tip and lipid bilayer
[13] in an electrolytic media.
F
4R tip bilayer

e
d

Here, F is the force, R is the tip radius,  is the Debye screening length, tip and
bilayer are tip and bilayer charge densities,  is the dielectric constant of the
medium and d is the distance between the tip and bilayer.
(b) Using the numerical solutions of non-linear Poisson-Boltzmann equation to obtain
the bilayer charge density. The numerical analysis will be carried in collaboration
with Dr. Scott R. Crittenden and Dr. Yuriy Pershin at University of South
Carolina, USA.
The results obtained by both the methods will be compared to judge the effectiveness of the
analytical expressions developed using the simplified models of AFM tip–sample interactions
and the numerical methods. Depending on the limitations found in either of the methods, we
will be in a position to choose an appropriate method for the future studies.
The effect of bilayer potential and ions in an electrolytic medium on the interaction
forces between the AMP molecules and the bilayer will be studied by measuring the electrical
double layer forces between the AMP molecules and the lipid bilayer. Again, AFM will be
employed to obtain the force-distance curves and the data will be analyzed to get the double
layer forces. In addition to the long range interaction forces between the lipid bilayer and
AMP molecules, these results also provide the information on the energy barrier that has to
be overcome by AMP molecules before binding to the lipid bilayer. The latter information is
important to understand the kinetics of AMP-membrane binding in the presence of various
ions in the medium.
Significance of the proposal and prospects of future work:
Our studies on the electrical interactions between AMPs and lipid bilayers
compliment the efforts being taken by many other research groups around the world to
understand and build AMPs with desirable applications. Currently, techniques of sum
frequency generation spectroscopy and NMR are being employed by other groups to
understand the structure and activity of AMP molecules under physiological conditions.
These complimentary studies together with the outcome of this proposed research will help to
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Bharat Kumar
get a clear picture of AMP-lipid bilayer interactions and build more accurate models to
describe the action of AMPs on target cells. Understanding the effect of structure, charge, etc.
of AMPs on the cell membranes will also help in engineering the AMP properties in order to
have applications like antibiotics [15,16] and other device applications like biosensors [17],
microbial resistant surfaces [18] etc.
The knowledge and experience gained by this study will help in continuing our study
on more complex systems like interactions between AMP and native cell membrane.
Comparing the outcome of this proposed research with the studies carried out on DNAmembrane interactions will also help in building a broader picture describing electrical
interactions between a polyelectrolyte and charged liquid crystalline membrane. In addition,
the expertise gained from this research work will help us to expand our work to other related
fields which may demand the probing of molecular-level forces.
[1]. Michael Zasloff; Nature, 415, 389 (2002).
[2]. Vitor, T., Maria J. Feio, Margarida, B.; Progress in Lipid Research, 51, 149 (2012).
[3]. Mihajlovic, M., Lazaridis, T.; Biochim. Biophys. Acta, 1798, 1494 (2010).
[4]. Jean-Francois, F., Elezgaray, J., Berson, P., Vacher, P., Dufourc, E.J.; Biophys. J., 95, 5748
(2008).
[5]. Wu, G., Wu, H., Li, L., Fan, X., Ding. J., Li,X.; Biochem. Biophys. Res. Commun., 395, 31
(2010).
[6]. Bond, P. J., Parton, D. L., Clark, J. F., Sanson, M. S.; Biophys. J., 95, 3802 (2008).
[7]. Muller, D.; Biochemistry, 47, 7986 (2008).
[8]. Castellana, E. T., Cremer, P. S.; Surf. Sci. Rep., 61, 429 (2006).
[9]. Muller, D. J., Engel, A.; Curr. Opin. Colloid Interface Sci., 13, 338 (2008).
[10]. Cappella, B., Dietler, G.; Surf. Sci. Rep., 34, 1 (1999).
[11]. Raiteri, R., Martinoia, S., Grattarola, M.; Biosensors and Bioelectronics, 11, 1009 (1996).
[12]. Sotres, J., Baro., A. M.; Biophys. J., 98, 1995 (2010).
[13]. Butt, H. J.; Biophys. J., 60, 777 (1991).
[14]. Yang, Y., Mayer, K. M., Hafner, J. H.; Biophys. J., 92, 1966 (2007).
[15]. Epand, R. F., Mor, A., Epand, R. M.; Cell. Mol. Life Sci., 68, 2177 (2011).
[16]. Vaara, M.; Current Opinion in Pharmacology, 9, 571 (2009).
[17]. Mannoor, M. S., Zhang, S., Link, A. J., McAlpine, M. C.; Proc. Natl. Acad. Sci. USA, 107,
19207 (2010).
[18]. Yala, J., Thebault, P., Hequet, A., Humblot, V., Pradier, C., Berjeaud, J.; Appl. Microbiol.
Biotechnol., 89, 623, (2011).
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