Abstract
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 known to be over-expressed in early stages of the disease. We designed and built Langmuir-Blodgett nanostructures incorporating ligands with high affinity for FKBP12: Tacrolimus (FK506) and Rifaximin as candidate nanosensors to detect low FKBP12 concentration in the initial phase of the amyloidosis. The binding process of the different ligands has been studied by means of photophysical measurements investigating the fluorescence quenching of the tryptophan residue in the binding pocket of FKBP12 by addition of the ligand in solution. Immobilization of the ligands was achieved adopting biomimetic strategy: phospholipid Langmuir-Blodgett films are proposed as nanoscaffolds for ligand inclusion. Several phospholipid nanoarchitectures differing in lipid composition, fluidity, number of layers and method of production (incubation versus co-spreading) were screened. The results have shown that both FK506 and Rifaximin ligands penetrate the lipid matrix either as monomers or as aggregates depending on their initial concentration. More importantly, the experiments demonstrated that the ligands in the LB scaffolds efficiently quench FKBP12 fluorescence in solution as a consequence of ligand binding to the protein.
Highlights
The FK506 binding proteins (FKBP) belong to a family of immunophilins with domains of protein-protein interactions and various cellular functions
We examined two ligands of FKBP12, namely FK506, a natural ligand known as Tacrolimus, and Rifaximin, an antibiotic molecule selected for its analogy of cyclic rigid structure with FK506
The binding process of the ligands was preliminary studied by means of photophysical measurements in solution investigating the fluorescence quenching of the tryptophan residue in the binding pocket of FKBP12 [6]
Summary
The FK506 binding proteins (FKBP) belong to a family of immunophilins with domains of protein-protein interactions and various cellular functions. Investigation on the interaction mechanism of this class of ligands with FKPB12 in the presence of candidates for fibrillization, i.e. α-synuclein, will provide an effective mean for elucidating the role of FKBP12 itself in amyloidogenesis [2,3] Results in these directions will be presented in forthcoming studies. The action mechanism of these drugs is based on the inhibition of RNA synthesis and MRP (Multidrug Resistance-associated Protein) activity responsible for the failure of anti-cancer therapies [4] Fluorescence quenching of FKBP12 in the presence of LB scaffold with immobilized ligands was studied, the results enabled to assess the occurrence of a binding process at the outer layer of the LB nanostructures
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