1
Simultaneous AFM, FS and X-Ray Reflectometry study of receptor- independent interactions of small-molecules with model lipid membranes. B. Gumí-Audenis 1,2,3,4 , F. Carlá 2 , A. Panzarella 2 , F. Comin 2 , F. Sanz 1,3,4 , L. Costa 2 and M.I. Giannotti 1,3,4 1 Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; 2 European Synchrotron Radiation Facility (ESRF), Grenoble (France); 3 Physical Chemistry Department, Universitat de Barcelona, Barcelona, Spain; 4 CIBER 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. Pure DPPC DPPC:Mel (95:5) Preliminary tests show that the mean rupture force value (F b ) of DPPC bilayers decreases after the addition of 5% of melatonin into the membrane. 15nN 10 5 0 Force F b (Pure DPPC) = 14,1 ± 1,0 nN F b (DPPC with 5% of Mel) = 5,7 ± 0,5 nN

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Page 1: Simultaneous AFM, FS and X-Ray Reflectometry study of ...€¦ · Simultaneous AFM, FS and X-Ray Reflectometry study of receptor-independent interactions of small-molecules with model

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