Alberto Ceccon

High Relaxivity Supramolecular Adducts Between Human‐Liver Fatty‐Acid‐Binding Protein and Amphiphilic GdIII Complexes: Structural Basis for the Design of Intracellular Targeting MRI Probes

Gadolinium complexes linked to an apolar fragment are known to be efficiently internalized into various cell types, including hepatocytes. Two lipid-functionalized gadolinium chelates have been investigated for the targeting of the human liver fatty acid binding protein (hL-FABP) as a means of increasing the sensitivity and specificity of intracellular-directed MRI probes. hL-FABP, the most abundant cytosolic lipid binding protein in hepatocytes, displays the ability to interact with multiple ligands involved in lipid signaling and is believed to be an obligate carrier to escort lipidic drugs across the cell. The interaction modes of a fatty acid and a bile acid based gadolinium complex with hL-FABP have been characterized by relaxometric and NMR experiments in solution with close-to-physiological protein concentrations. We have introduced the analysis of paramagnetic-induced protein NMR signal intensity changes as a quantitative tool for the determination of binding stoichiometry and of precise metal-ion-center positioning in protein-ligand supramolecular adducts. A few additional NMR-derived restraints were then sufficient to locate the ligand molecules in the protein binding sites by using a rapid data-driven docking method. Relaxometric and (13)C NMR competition experiments with oleate and the gadolinium complexes revealed the formation of heterotypic adducts, which indicates that the amphiphilic compounds may co-exist in the protein cavity with physiological ligands. The differences in adduct formation between fatty acid and bile acid based complexes provide the basis for an improved molecular design of intracellular targeted probes.

Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 μs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 μs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.

NMR investigation of the equilibrium partitioning of a water‐soluble bile salt protein carrier to phospholipid vesicles

Membrane binding by cytosolic fatty acid binding proteins (FABP) appears to constitute a key step of intracellular lipid trafficking. We applied NMR spectroscopy to study the partitioning of a water‐soluble bile acid binding protein (BABP), belonging to the FABP family, between its free and lipid‐vesicle‐bound states. As the lipid‐bound protein was NMR‐invisible, the signals of the free biomolecule were analyzed to obtain quantitative information on binding affinity and steady‐state kinetics. The data indicated a reversible interaction of BABP with anionic vesicles occurring in a very slow exchange regime on the NMR time scale. The approximate binding epitope was demonstrated from results on BABP samples in which different positively charged lysine residues were mutated to neutral alanines. H/D exchange measurements indicated a higher exposure to solvent for the core amino acid residues in the liposome‐bound state. Finally, the BABP‐liposome interaction was also investigated for the first time through an MRI‐chemical exchange saturation transfer experiment that has potential applications not only in the field of biology, but also in biomedicine, bioanalytical chemistry, and nanotechnology. Proteins 2013; 81:1776–1791.

Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin

The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ubs NMR signals, together with (15)N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.

Dynamics of a globular protein adsorbed to liposomal nanoparticles.

A solution-state NMR method is proposed to investigate the dynamics of proteins that undergo reversible association with nanoparticles (NPs). We applied the recently developed dark-state exchange saturation transfer experiment to obtain residue-level dynamic information on a NP-adsorbed protein in the form of transverse spin relaxation rates, R2bound. Based on dynamic light scattering, fluorescence, circular dichroism, and NMR spectroscopy data, we show that the test protein, human liver fatty acid binding protein, interacts reversibly and peripherally with liposomal NPs without experiencing significant structural changes. The significant but modest saturation transfer from the bound state observed at 14.1 and 23.5 T static magnetic fields, and the small determined R2bound values were consistent with a largely unrestricted global motion at the lipid surface. Amino acid residues displaying faster spin relaxation mapped to a region that could represent the epitope of interaction with an extended phospholipid chain constituting the protein anchor. These results prove that atomic-resolution protein dynamics is accessible even after association with NPs, supporting the use of saturation transfer methods as powerful tools in bionanoscience.

Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects.

Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP–lysozyme complex was obtained by data‐driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment.

The unique ligand binding features of subfamily-II iLBPs with respect to bile salts and related drugs ☆

Intracellular lipid binding proteins (iLBPs) are a family of evolutionarily related small cytoplasmic proteins implicated in the transcellular transport of lipophilic ligands. Subfamily-II iLBPs include the liver fatty acid binding protein (L-FABP), and the ileal and the liver and ileal bile acid binding proteins (L-BABP and I-BABP). Atomic-level investigations during the past 15-20 years have delivered relevant information on bile acid binding by this protein group, revealing unique features including binding cooperativity, promiscuity, and site selectivity. Using NMR spectroscopy and other biophysical techniques, our laboratories have contributed to an understanding of the molecular determinants of some of these properties and their generality among proteins from different animal species. We focused especially on formation of heterotypic complexes, considering the mixed compositions of physiological bile acid pools. Experiments performed with synthetic bile acid derivatives showed that iLBPs could act as targets for cell-specific contrast agents and, more generally, as effective carriers of amphiphilic drugs. This review collects the major findings related to bile salt interactions with iLBPs aiming to provide keys for a deeper understanding of protein-mediated intracellular bile salt trafficking.

Probing the Binding Modes of a Multidomain Protein to Lipid-based Nanoparticles by Relaxation-based NMR

The interactions of two model multidomain proteins-covalently linked diubiquitins, Ub2-with lipid-based nanoparticles have been quantitatively probed by the measurements of NMR lifetime line broadening, ΔR2. By combined analysis of ΔR2 profiles arising from interactions with liposomes of varying sizes, an approach recently developed for the characterization of interactions of monoubiquitin with liposomes, we determine how the parameters of exchange (liposome binding) and dynamics of each individual domain of Ub2 on the surface of liposomes change when the domains are covalently attached to one another by a flexible linker. Two different covalent linkages were used: K63-linked and K48-linked Ub2. The possibility of three distinct modes of binding of Ub2 to liposomes requires the introduction of simple but important modifications to the strategy of analysis originally developed for monoubiquitin.

Are they in or out? The elusive interaction between Qtracker(®)800 vascular labels and brain endothelial cells

AIM Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.