Nanolayers for diagnostics of proteins

involved in degenerative amyloidosisM. R. Martina and G. Caminati

Department of Chemistry/CSGI, University of Florence, via della Lastruccia, 3, I-50019, Sesto Fiorentino (FI), Italy

e-mail: martina_m@csgi.unifi.it

FK-506 binding protein (FKBP12) is a protein of the family of immunophilins, involved in many neurodegenerative diseases such as

Alzheimer’s syndrome where FKBP12 is found to be overexpressed in early stages of the disease. We design and built Langmuir-

Blodgett nanostructures incorporating ligands with high affinity for FKBP12: Tacrolimus (FK506) and Rifaximin as possible

nanosensors to detect low FKBP12 concentration in the initial phase of the amyloidosis. In particular, we examined Langmuir-

Blodgett (LB) mono and bilayers, incorporating the ligand either by immersion of the lipid layers in the ligand solution or by co-

spreading in the preparation of the precursor monolayer.

Introduction

The binding process of the ligands was preliminary studied by means of photophysical measurements investigating the fluorescence quenching of the tryptophan residue in the binding pocket of FKBP12. Kd values for Rapamycin was used as standard.

• The incorporation of ligands and their interaction with FKBP12

in biomimetic models opens the possibility of developing sensitive

and specific nanosensors for FKBP12 for the diagnosis of

neurodegenerative amyloidosis in the early phase

• Other potential ligands with different functional groups will be

tested to identify the most effective compound and whose

inclusion in the bilayers is most favored. The efficient ligands will

be functionalized with suitable spacers for anchoring to surfaces

• Formation of beta-amyloid with model proteins will be

monitored with free and ligand bound FKBP12 to assess the

therapeutic effect of the FKBP12 inhibition.

FK506

Rifaximin

5.5 ± 0.2Rapamycin

13.8± 0.3Rifaximin

6.2± 0.2FK506

Kd

(nM)Ligands

By addition of the

ligand in solution

Efficient fluorescence

quenching of FKBP12

Decreasing intensity of FKBP12 relative

fluorescence with FK506 (squares), Rifaximin

(triangles) and Rapamycin (circles)

Increasing

concentration of

ligand

Photophysical study of FKBP12-ligands binding

Inclusion of ligands in biomimetic models

Increasing concentration of FK506 π=25mN/m

Increasing concentration of FK506 π=35mN/m

Increasing concentration of FK506 π=45mN/m

Fluorescence excitation (dotted line) and

emission (solid line) spectra of LB incubated in

[Rfx]= 1x10-5M (red), [Rfx]= 0.8x10-5M (green),

[Rfx]= 1x10-6M (black). λexc=450nm and

λem=620nm. Inset: UV-Vis spectra of LB

incubated in Rfx.

Change in the mean molecular area (∆A) with respect to the initial

molecular area of DPPC/POPG in the absence of FK506 (A0) at

constant surface pressure (π=32 mN m-1) as a function of mole

percentage of FK506 in FK506/DPPC/POPG mixed monolayers at

25 °C. ). Inset: fluorescence emission spectra of FK506 in 1LB layer

of DPPC/POPG 8:2.

LB of DPPG +Rfx included

by incubation

LB of DPPC/POPG + FK506 included

by co-spreadingLigand Phospholipid

Incubation

1 LB Ligand solution

Mixed monolayer of

DPPC/POPG/FK506

BAM images of DPPC/POPG monolayers with

increasing concentration of FK506 at different surface

pressure on TRIS subphase at 25°C.

Both Rfx and FK506 penetrates easily the lipid matrix and we safely exclude migration of the ligand from the

monolayer to the subphase also in the condensed phase chosen for LB transfer.

Ligand

FKBP12

FKBP12-ligands interaction in LB films25

20

15

10

5

Flu

ore

sce

nce

In

ten

sity

370360350340330320

Wavelength, nm

Quenching

+ RFX

+ FK506

No ligands

Fluorescence emission spectra of: Rfx in LB pre-incubation with FKBP12 1μM (blue), Rfx in LB

during incubation with FKBP12 1μM (green), Rfx in LB post-incubation with FKBP12 1μM (black).

Blueshift of major peak of Rfx

and band appearance in

LB+Rfx+FKBP at about 650nm

100

80

60

40

20

Norm

aliz

ed F

luo

rescence

Inte

nsity

800750700650600550500

Wavelength, nm

λexc=450nm

Change of Rifaximin

molecular

microenvironment

Efficient fluorescence

quenching of FKBP

with both ligands.

Higher quenching was

found for the Rfx

ligand as result

related to the larger

ligand concentration

obtained with the

incubation procedure.

Conclusions

M.R.M acknowledges MIUR (FIRB RBPR05JH2P Itananonet) for financial support.

Acknowledgements