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Simultaneous AFM, FS and X-Ray Reflectometry study of receptor-
independent interactions of small-molecules with model lipid membranes. B. Gumí-Audenis1,2,3,4, F. Carlá2, A. Panzarella2, F. Comin2, F. Sanz1,3,4, L. Costa2 and M.I. Giannotti1,3,4
1Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; 2European Synchrotron Radiation Facility (ESRF), Grenoble (France); 3Physical Chemistry Department, Universitat de Barcelona, Barcelona, Spain; 4CIBER de Bioingeniería, Biomateriales y Nanomedicina
(CIBER-BBN), Madrid, Spain. [email protected]
The Atomic Force Microscope (AFM) is an excellent instrument to characterize surfaces at the nanoscale at the single molecule level. Moreover,
AFM-based Force Spectroscopy (FS) is essential to probe local properties of Supported Lipid Bilayers (SLBs) in the direct space. Although having a
high spatial range sensitivity and versatility, the lateral and vertical resolution obtained by AFM might be inferior to the X-Ray techniques
depending on the density, the order and the composition of the sample. Accordingly, X-Ray Reflectometry (XRR) is a powerful tool to characterize
surfaces at nanoscale, yielding the vertical structural information of the specimen in the reciprocal space.
AFM imaging: Morphology FS: Nanomechanics XRR: Vertical structure
Custom AFM for X-Ray end-stations
We have developed a fast AFM which
can be integrated as a synchrotron
radiation sample holder for “grazing-
incidence” in-situ X-Ray experiments.
X-AFM in ID03 (ESRF)
Induced sample changes (i.e.
phase transitions and chemical
reactions)
Radiation damage
Alignment of nano-objects
Radiation damage on phospholipid bilayers
In both experiments, a decrease in intensity was observed in XRR
curves leading to a complete disappearance of the fringes
AFM image after full beam exposure
AFM image before XRR
AFM image after 1 XRR
DOPC bilayers: Material deposition on top of the
membranes after 1 XXR
Membrane disappearance after
full beam exposure
AFM image after 1 XRR
AFM image before XRR
DPPC bilayers: Holes formation in the membrane structure after 1 XXR
1. B. Gumí-Audenis, F. Carlá, M.V. Vitorino, A. Panzarella, L. Porcar, M.Boilot, S. Guerber, B. Pascal, M.S. Rodrigues, F. Sanz, M.I. Giannotti and L. Costa. Custom AFM for X-Ray beamlines: in-situ biological
investigations under physiological conditions. Currenly accepted in Journal of Synchrotron Radiation, 22, 2015.
2. B. Gumí-Audenis, F. Sanz and M.I. Giannotti. Impact of Galactosylceramide on the nanomechanical properties of lipid bilayer models: AFM-force spectroscopy study. Soft Matter, 11, 5447-5454, 2015.
3. L. Redondo-Morata, M.I. Giannotti and F. Sanz. Stability of Lipid Bilayers as Model Membranes: Atomic Force Microscopy and Spectroscopy Approach. In Atomic Force Microscopy in Liquid. Edited by
Baró AM, Reifenberger RG: Wiley-VCH Verlag GmbH & Co.KGaA; 2012.
4. Evers, F. C. Jeworrek, K. Weise, M. Tolan and R. Winter. Detection of lipid raft domains in neutral and anionic Langmuir monolayers and bilayers of complex lipid composition. Soft Matter, 8 (7), 2170-
2175, 2012.
References
Biological membranes and small-molecules
Biological membranes: Permeability barriers for cells and organelles
Structural role under a combination of forces
Phospholipid bilayers can be used as membrane
model systems due to their resemblance.
First line of defense against invading species
Their chemical composition and their non-specific interaction with small
molecules can alter their structure and physical properties, affecting their
interaction with other surrounding molecules, i.e. peptides.
Supported Lipid Bilayer (SLB)
Melatonin (Mel), a small-molecule derived from Trp,
has been recently linked to roles that involve non-
specific and receptor-independent interactions
with the lipid membrane. Mel has protective effects
in several diseases and seems to influence to the
membrane fluidity.
Tryptophan (Trp) exerts its effect through the specific binding to the membrane receptors.
Pu
re D
PP
C
DP
PC
:Me
l (9
5:5
)
Preliminary tests show that the mean rupture force value (Fb) of DPPC
bilayers decreases after the addition of 5% of melatonin into the
membrane.
15nN
10
5
0
Fo
rce
40nm200-20
Sep
Fb (Pure DPPC) = 14,1 ± 1,0 nN
Fb (DPPC with 5% of Mel) = 5,7 ± 0,5 nN