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Rhodopsins are photoreceptive proteins using light to drive a plethora of biological functions such as vision, proton and ion pumping, cation and anion channeling, and gene and enzyme regulation. Here we combine organic synthesis, NMR structural studies, and photochemical characterization to show that it is possible to prepare a fully synthetic mimic of rhodopsin photoreceptors. More specifically, we conjugate a bile acid binding protein with a synthetic mimic of the rhodopsin protonated Schiff base chromophore to achieve a covalent complex featuring an unnatural protein host, photoswitch, and photoswitch-protein linkage with a reverse orientation. We show that, in spite of its molecular-level diversity, light irradiation of the prepared mimic fuels a photochromic cycle driven by sequential photochemical and thermal Z/E isomerizations reminiscent of the photocycles of microbial rhodopsins.

Electrospinning is here used for the first time to prepare nanofibers including a host/guest complex in a keratin/poly(ethylene oxide) matrix. The host is a lipid binding protein and the guest is an insoluble bactericidal molecule, irgasan, bound within the protein internal cavity. The obtained nanofibers, characterized by scanning electron microscopy, exhibit excellent antibacterial activity toward Gram positive and negative bacteria, even with a moderate protein/irgasan cargo. Solution NMR studies, employed to provide molecular information on the cargo system, points to a micromolar affinity, compatible with both the electrospinning process and slow guest release. The versatility of the carrier protein, capable of interacting with a variety of druggable hydrophobic molecules, is exploitable for the development of innovative biomedical devices, whose properties can be tuned by the selected guest., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off., (© 2015 FEBS.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

New strategies are requested for the preparation of bioinspired host-guest complexes to be employed in technologically relevant applications, as sensors and optoelectronic devices. We report here a new approach employing a single monomeric protein as host for the strongly fluorescent rhodamine dye. The selected protein, belonging to the intracellular lipid binding protein family, fully encapsulates one rhodamine molecule inside its cavity forming a host-guest complex stabilized by H and π-hydrogen bonds, a salt bridge, and favorable hydrophobic contacts, as revealed by the NMR derived structural model. The protein-dye solutions are easily processable and form homogeneous thin films exhibiting excellent photophysical and morphological properties, as derived from photoluminescence and AFM data. The obtained results represent the proof of concept of the viability of this bio host-guest system for the development of bioinspired optoelectronic devices.

The presence of a disulfide bridge in liver bile acid binding protein (L-BABP/S-S) allows for site-selective binding of two bile acids, glycochenodeoxycholic (GCDA) and glycocholic acid (GCA), differing only in the presence of a hydroxyl group. The protein form devoid of the disulfide bridge (L-BABP) binds both bile salts without discriminating ability. We investigate the determinants of the molecular recognition process in the formation of the heterotypic L-BABP/S-S complex with GCDA [corrected] and GCA [corrected] located in the superficial and inner protein sites, respectively. The comparison of the NMR spectroscopy structure of heterotypic holo L-BABP/S-S, the first reported for this protein family, with that of the homotypic L-BABP complex demonstrates that the introduction of a S-S link between adjacent strands changes the conformation of three key residues, which function as hot-spot mediators of molecular discrimination. The favoured χ(1) rotameric states (t, g(+) and g(-) for E99, Q100 and E109 residues, respectively) allow the onset of an extended intramolecular hydrogen-bond network and the consequent stabilisation of the side-chain orientation of a buried histidine, which is capable of anchoring a specific ligand., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

Bile acid-conjugated gadolinium chelates were shown to display promising features for the development of hepatospecific constrast agents for magnetic resonance imaging (MRI). The study of the pharmacokinetics of these compounds should address their possible interaction with the bile acid protein transporters. We have previously shown that a 5beta-cholanoic acid-based contrast agent is efficiently internalized in hepatocytes and is able to bind to a liver bile acid binding protein (BABP) in vitro. Here we report the solution structure of the adduct between a BABP and a gadolinium chelate/bile acid conjugate. The identification of unambiguous intermolecular distance restraints was possible through 3D edited/filtered NOESY-HSQC experiments, together with distance information derived from paramagnetic relaxation enhancements. These intermolecular contacts were used for the structure determination of the complex, using the data-driven docking software HADDOCK. The obtained results represent the starting point for the design of new and more efficient MRI contrast agents.

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family., ((c) 2008 Wiley-Liss, Inc.)

It has been proposed that intracellular carrier proteins mediate active transport of the bile acids within hepatocytes and ileocytes, during the enterohepatic circulation. In mammalian species only ileal bile acid binding proteins have been so far identified, while liver cytosolic carriers have never been found. On the contrary, in non-mammalian vertebrates, only liver, and not ileal, bile acid binding proteins were reported. The aim of the present work is to find the missing cytosolic transport proteins. A bioinformatic search allowed us to identify a non-mammalian putative bile acid binding protein in the chicken ileum (cI-BABP), which we recombinantly expressed and purified. The protein exhibits the capability, tested by in vitro NMR experiments, of binding bile acids. Furthermore, strong NMR evidence reported that the human liver fatty acid binding protein (hL-FABP) can also bind bile acids. Taken together, these data strongly suggest that both cI-BABP and hL-FABP have a bile acid binding function in the two organisms, and support a previous hypothesis on the role of hL-FABP in regulating bile acid metabolism and determining bile acid pool size., ((c) 2007 Wiley-Liss, Inc.)

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.

The binding affinities of a selected series of Gd(III) chelates bearing bile acid residues, potential hepatospecific MRI contrast agents, to a liver cytosolic bile acid transporter, have been determined through relaxivity measurements. The Ln(III) complexes of compound 1 were selected for further NMR structural analysis aimed at assessing the molecular determinants of binding. A number of NMR experiments have been carried out on the bile acid-like adduct, using both diamagnetic Y(III) and paramagnetic Gd(III) complexes, bound to a liver bile acid binding protein. The identified protein "hot spots" defined a single binding site located at the protein portal region. The presented findings will serve in a medicinal chemistry approach for the design of hepatocytes-selective gadolinium chelates for liver malignancies detection.

Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions., (2007 Wiley-Liss, Inc.)

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.