2P070
Probing Interaction of a Cell Penetrating Peptide with Lipid at Air/Water Interface Using
Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy(Molecular Spectroscopy Laboratory, RIKEN) ADHIKARI, Aniruddha; NIHONYANAGI,
Satoshi; YAMAGUCHI, Shoichi; TAHARA, Tahei[Introduction]
Treatment of diseases often involves delivery of therapeutic molecules into cells. Thus,
transportation of bio-molecules across cell membranes is an issue of utmost biological importance. The
efficiency of such a process is governed by the interaction between the therapeutic molecule and the lipid
bilayer of the cell membrane. Short-chain, basic amino acid-rich sequences, commonly known as Cell
Penetrating Peptides (CPPs) are a class of molecules that facilitate this phenomenon.1 However, amolecular level mechanism that describes this interfacial transport is currently lacking since suchinterfacial regions are often only a few nanometers in thickness and are difficult to investigate.
Heterodyne-detected Vibrational Sum Frequency Generation (HD-VSFG) is a novel nonlinear
spectroscopic tool which allows us to selectively monitor molecular process occurring at the interface.2,3
In particular, the HD-VSFG experiment is capable of determining the net orientation of water molecules
at various interfaces. The sign of the OH band as revealed in the imaginary χ(2) spectrum of interfacial
water provides a direct evidence about whether the water molecule is oriented with its H atoms pointing
upwards or away from the interface.2,3
In this presentation, we report a HD-VSFG study that aims to unravel the interaction of a
prototypical CPP molecule with a model lipid monolayer comprising of an anionic lipid.
[Experiment]
We chose deuterated 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, sodium salt (d-DPPG) as themodel lipid for our study. It belongs to the biologically important class of membrane phospholipids and it
is anionic in nature. Figure 1 shows the chemical structure of d-DPPG.
Figure 1. Chemical structure of deuterated 1,2dipalmitoyl-sn-glycero-3-phosphoglycerol (d-DPPG).

For CPP, we used an arginine oligomer (octa-arginine) in which the N-terminal was modified with astearyl chain and its C-terminal amidified. Such modification has been reported to enhance the cellular
uptake of cargo molecules quite significantly.4 Thus stearyl octa-arginine (henceforth abbreviated as SR8)was used in our studies as a prototype to represent the behavior of CPPs. Figure 2 shows the chemical
formula for the primary structure of SR8.
CH3-(CH2)16-CONH-RRRRRRRR-CONH2 where R= arginine =Figure 2. Chemical formula for Stearyl Octa-arginine

All measurements were carried out for lipid monolayer at the air/water interface with the subphaseconsisting of 10 mM Tris.HCl buffer solution (pH 7.5) at 23 oC. For our HD-VSFG experiments, thesample was excited simultaneously with a pair of pulses: i) a broadband infrared pulse (ω2) containing
frequencies in resonance with the vibrational transitions of interest and ii) a narrowband visible pulse (ω1).
The sum frequency generated from the sample was subsequently mixed with sum frequency light from a
local oscillator to achieve heterodyne detection. The sum frequency, visible and infrared beams were s-, sand p-polarized (ssp polarization) respectively, in the present work.
[Results and Discussion]
The interaction between the anionic negativelyA

charged deuterated DPPG lipid interface and the arginine

electrostatic attraction. On addition of SR8 to the d-DPPG
monolayer, we observe a change in the sign of the OHband (above 3000 cm-1) for interfacial water (Figure 3).
This can be attributed to the modification of the lipidIm χ(2) (a.u.)rich positively charged CPP is expected to be governed byB

interface upon CPP adsorption. The net charge of theinterface goes from negative to positive, causing a flip of
the adjacent interfacial water molecules. The present
study clarifies that water molecules are initially orientedω2 (cm-1)

with their H atoms up and re-orient themselves with their
H atoms pointed downwards following adsorption of theFigure 3. Im χ(2) spectra of air/water interface

positively charged CPP.

with A) d- DPPG and B) after addition of 0.5
μM of SR8 peptide.

[References]
1. Hoyer, J. ; Neundorf, I. Acc. Chem. Res. 2012, 45 (7) 1048.
2. Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. J. Am. Chem. Soc. 2010, 132, 10656.
3. Mondal, J. A.; Nihonyanagi, S.; Yamaguchi, S.; Tahara, T. J. Am. Chem. Soc. 2012, 134, 7842.
4. Khalil, I. A.; Futaki, S.; Niwa, M.; Baba, Y.; Kaji, N.; Kamiya, H.; Harashima, H. Gene Thereapy
2004, 11, 636.