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: [email protected]
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
Recommended
