Bile Acid Molecules Research Articles

Cholic Acid-Derived Gemini Amphiphile Can Eradicate Interkingdom Polymicrobial Biofilms and Wound Infections.

Biofilm infections are mainly caused by Gram-positive bacteria (GPB) like Staphylococcus aureus, Gram-negative bacteria (GNB) like Pseudomonas aeruginosa, and fungi like Candida albicans. These infections are responsible for antimicrobial tolerance, and commensal interactions of these microbes pose a severe threat to chronic infections. Treatment therapies against biofilm infections are limited to eradicating only 20-30% of infections. Here, we present the synthesis of a series of bile acid-derived molecules using lithocholic acid, deoxycholic acid, and cholic acid where two bile acid molecules are tethered through 3'-hydroxyl or 24'-carboxyl terminals with varying spacer length (trimethylene, pentamethylene, octamethylene, and dodecamethylene). Our structure-activity relationship investigations revealed that G21, a cholic acid-derived gemini amphiphile having trimethylene spacer tethered through the C24 position, is a broad-spectrum antimicrobial agent. Biochemical studies witnessed that G21 interacts with negatively charged lipoteichoic acid, lipopolysaccharide, and phosphatidylcholine moieties of GPB, GNB, and fungi and disrupts the microbial cell membranes. We further demonstrated that G21 can eradicate polymicrobial biofilms and wound infections and prevent bacteria and fungi from developing drug resistance. Therefore, our findings revealed the potential of G21 as a versatile antimicrobial agent capable of effectively targeting polymicrobial biofilms and wound infections, suggesting that it is a promising antimicrobial agent for future applications.

Natural Dual‐Crosslinked Self‐Healing Hydrogels for In Situ Wound Healing

AbstractPoly(l‐glutamic acid) (PLGA), a polypeptide prepared by bacteria fermentation, has broad applications in tissue engineering. A natural host–guest pair of β‐cyclodextrin (β‐CD) and bile acid molecules are attached covalently onto the PLGA backbones separately, and host–guest crosslinked self‐healing hydrogels can be easily formed by mixing these polymer solutions. Inspired by the adhesives of mussel foot proteins, a dopamine methyl acrylamide derivative is introduced into this system to prepare a PLGA‐based dual‐network hydrogel in situ through the combination of host–guest crosslinking and the photo‐crosslinking of the dopamine derivative. The mechanical properties of the hydrogels can be tuned by altering the molar ratios of β‐CD and bile acid as well as the content of dopamine methyl acrylamide derivative. The dynamic inclusion complexation of β‐CD and bile acid allows the hydrogel to self‐heal rapidly under ambient atmosphere and their mechanical properties can recover to original levels within 1 min as evidenced in rheological results. The dual‐network hydrogels exhibit stronger adhesion behavior to pig skin than the host–guest crosslinked hydrogels in vitro. The adhesion of the dual‐network hydrogels to rat whole cortical incision has promoted faster wound healing and tissue regeneration than that in sutured treatments in vivo.

Identification and Spatial Visualization of Dysregulated Bile Acid Metabolism in High-Fat Diet-Fed Mice by Mass Spectral Imaging.

Changes in overall bile acid (BA) levels and specific BA metabolites are involved in metabolic diseases, gastrointestinal, and liver cancer. BAs have become established as important signaling molecules that enable fine-tuned inter-tissue communication within the enterohepatic circulation. The liver, BAs site of production, displayed physiological and functional zonal differences in the periportal zone versus the centrilobular zone. In addition, BA metabolism shows regional differences in the intestinal tract. However, there is no available method to detect the spatial distribution and molecular profiling of BAs within the enterohepatic circulation. Herein, we demonstrated the application in mass spectrometry imaging (MSI) with a high spatial resolution (3 μm) plus mass accuracy matrix-assisted laser desorption ionization (MALDI) to imaging BAs and N-1-naphthylphthalamic acid (NPA). Our results could clearly determine the zonation patterns and regional difference characteristics of BAs on mouse liver, ileum, and colon tissue sections, and the relative content of BAs based on NPA could also be ascertained. In conclusion, our method promoted the accessibility of spatial localization and quantitative study of BAs on gastrointestinal tissue sections and demonstrated that MALDI-MSI was a valuable tool to investigate and locate several BA molecules in different tissue types leading to a better understanding of the role of BAs behind the gastrointestinal diseases.

Prevention of the rise in plasma cholesterol and glucose levels by kaki-tannin and characterization of its bile acid binding capacity.

Bile acid-binding agents, such as cholestyramine and colesevelam, improve both cholesterol and glucose metabolism. Kaki-tannin, a polymerized condensed tannin derived from persimmon (Diospyros kaki), has been shown to have bile acid-binding capacity and a hypocholesterolemic effect. However, its effects on glucose metabolism have not been well studied, and the binding selectivity of kaki-tannin to bile acid molecules has not been reported. In vivo experiments using mice with high-fat diet-induced obesity showed that kaki-tannin intake (20 g kg-1 of the diet) increased fecal bile acid excretion by 2.3-fold and prevented a rise in plasma cholesterol levels and fasting plasma glucose levels. Kaki-tannin also suppressed the development of impaired glucose tolerance. To characterize the bile acid-binding capacity of kaki-tannin, we investigated its capacity to bind to eight types of bile acid and cholesterol in vitro. Kaki-tannin showed strong capacity to bind to lithocholic acid (85.5%), which has one hydroxy group. It also showed moderate capacity to bind to bile acids with two hydroxy groups (53.3%), followed by those with three hydroxy groups (39.0%), but kaki-tannin did not show binding capacity to cholesterol. These results suggest that the binding capacity of kaki-tannin to bile acids tends to decrease as the number of hydroxy groups increases. Interestingly, the binding capacity of kaki-tannin correlated with that of cholestyramine (correlation coefficient: r = 0.900). Our findings indicate that kaki-tannin binds preferentially to bile acids with fewer hydroxy groups and has beneficial effects on glucose metabolism as well as cholesterol metabolism. © 2020 Society of Chemical Industry.

Collision Cross Section Conformational Analyses of Bile Acids via Ion Mobility-Mass Spectrometry.

Bile acids serve as one of the most important classes of biological molecules in the gastrointestinal system. Due to their structural similarity, bile acids have historically been difficult to accurately annotate in complex biological matrices using mass spectrometry. They often have identical or nominally similar mass-to-charge ratios and similar fragmentation patterns that make identification by mass spectrometry arduous, normally involving chemical derivatization and separation via liquid chromatography. Here, we demonstrate the use of drift tube ion mobility (DTIM) to derive collision cross section (CCS) values in nitrogen drift gas (DTCCSN2) for use as an additional descriptor to facilitate expedited bile acid identification. We also explore trends in DTIM measurements and detail structural characteristics for differences in DTCCSN2 values between subclasses of bile acid molecules.

A novel voltammetric approach to the detection of primary bile acids in serum samples

An innovative voltammetric approach to the detection of cholic and chenodeoxycholic acids is presented. These two primary bile acids are important biomarkers of liver function in humans and are involved in many physiological processes in the human body. Herein we describe a way to reproducibly convert the hard-to-detect bile acid molecule into an easily detectable derivative in situ using 0.1 M HClO4 in acetonitrile (water content 0.55%). Under these conditions the bile acids are dehydrated and the resulting alkenes can be subsequently oxidized electrochemically on polished boron-doped diamond electrode under unchanged conditions at approximately +1.2 V vs. Ag/AgNO3 in acetonitrile. After optimization, differential pulse voltammetry provides competitive limits of detection of 0.5 µM and 1.0 µM for cholic and chenodeoxycholic acid, respectively, with a linear course of calibration dependency to the minimum of 80 µM. The method was applied for detection of cholic and chenodeoxycholic acids in artificial and human serum samples using single solid phase extraction on C-18 cartridge for preliminary separation of the analytes. High recoveries of 80–90% were consistently obtained by the proposed voltammetric method and reference HPLC with fluorescence detection for human serum samples, confirming good selectivity for real-life samples.

Lipid and Bile Acid Dysmetabolism in Crohn’s Disease

Crohn's disease is one of the systemic autoimmune diseases. It commonly affects the small intestine and colon but may involve any portion of the gastrointestinal tract from the mouth to the anus. The most affected area by Crohn's disease is the distal part of the small intestine, in which the bile acid molecules are most efficiently reabsorbed. Bile acids form mixed micelles together with fatty acids, which function as a transport vehicle to deliver fatty acids to the apical membrane of enterocytes for absorption. Therefore, if the terminal ileum is impaired, bile acid malabsorption may occur, which may cause congenital diarrhoea in Crohn's disease. Similarly, the impairment of the terminal ileum also induces fatty acid malabsorption, which may influence the role of fatty acids in Crohn's disease. In contrast, a recent study reported that multidrug resistance protein 1 (MDR1) regulated effector T-cell function in the ileum from bile acid-driven oxidative stress and MDR1 loss of function in a subset of patients with Crohn's disease. However, the role of consumption of fatty acids in Crohn's disease remains to be fully elucidated. This review is aimed at providing an overview of some recent developments in research of Crohn's disease from comprehensive perspective with a focus on the connection between disease location and behaviour, lipid diets, and bile acid malabsorption.

Photoperiodic Flower Mimicking Metallic Nanoparticles for Image-Guided Medicine Applications.

Nanoradiosensitizers have been developed to enhance localization and precision of therapeutic radiation delivery. A specific volume of comprising surface atoms is known to be the radiosensitizing region. However, the shape-dependent local dose enhancement of nanoparticles is often underestimated and rarely reported. Here, a noble metal nanostructure, inspired by the photoperiodic day-flowers, was synthesized by metal reduction with bile acid molecules. The impact of high surface area of day-flowermimicking metallicnanoparticles (D-NP) on radiosensitizing effect was demonstrated with assays for ROS generation, cellular apoptosis, and clonogenic survival of human liver cancer cells (HepG2) cells. In comparison with lower-surface-area spherical night-flower mimickingmetallicnanoparticles (N-NP), exposure of our D-NP to external beam radiation doses led to a significant increase in reactive oxygen species (ROS) production and radiosensitizing cell cycle synchronization, resulting in an enhanced cancer-cell-killing effect. In clonogenic survival studies, dose-enhancing factor (DEF) of D-NP was 16.5-fold higher than N-NP. Finally, we demonstrated in vivo feasibility of our D-NP as a potent nanoradiosensitizer and CT contrast agent for advanced image-guided radiation therapy.

Gallstone-Formation-Inspired Bimetallic Supra-nanostructures for Computed-Tomography-Image-Guided Radiation Therapy.

Inspired by the gallstone formation mechanism, we report a fast one-pot synthesis of high-surface-area bimetallic hierarchical supra-nanostructures. As gallstones are generated from metal cholate complexes, cholate bile acid molecules with Au/Ag metal precursors formed stable nanocomplexes aggregated with metal Au ions and preformed ~2 nm silver halide nanoparticles before reduction. When a reducing agent was added, the metal cholate nanocomplexes quickly formed noble bimetallic hierarchical supra-nanostructures. The morphology of bimetallic supra-nanostructures could be tailored by changing the feeding ratio of each metal precursor. In situ synchrotron small-angle X-ray scattering measurement with a custom-designed reaction cell showed two-step growth and attachment behavior toward hierarchical supra-nanostructures from the gallstone-formation-inspired metal cholate nanocomplexes in a 60 s reaction. Additional wide-angle X-ray scattering, X-ray absorption near-edge structure, in situ Fourier transform infrared, and high-resolution scanning transmission electron microscopy investigations subsequently revealed the mechanism for the evolution of bimetallic hierarchical supra-nanostructures. The gallstone-formation-inspired synthesis mechanism can be universally applied to other metals, for example, Pt-Ag and Pd-Ag bimetallic nanostructures. Finally, the synthesized high-surface-area bimetallic supra-nanostructures demonstrated significantly enhanced X-ray computed tomography imaging contrast and radiosensitizing effect for a potential image-guided nanomedicine application. We believe that our synthetic method inspired by gallstone formation and understanding represents an important step toward the development of hierarchical nanoparticles for various applications.

Structural and functional implications of the interaction between macrolide antibiotics and bile acids.

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC50) values.

Investigations on microstructural changes in pH responsive mixed micelles of Triton X-100 and bile salt

Aqueous solution behaviour of a nonionic surfactant Triton X-100 is investigated in the presence of two bile salts namely sodium deoxycholate (NaDC) and sodium cholate (NaC) at different pH, temperatures and in the presence of sodium chloride and the resultant structural changes to accordingly formed mixed micelles were analyzed by using cloud point (CP), viscosity and scattering techniques. Both the bile salts increased the CP and showed a corresponding decrease in viscosity and apparent hydrodynamic diameter (Dh), which can further be subsided with the progressive addition of sodium chloride and an increase in temperature. Interestingly, in the presence of bile salt below pH ∼5, CP decreased with corresponding increase in viscosity, while a reversed trend was observed above pH∼8. Small angle neutron scattering data reveal that nearly spherical mixed micelles were formed in the presence of bile salt which grow and transform to prolate ellipsoidal ones at pH∼3. These morphological changes are facilitated by the protonation of carboxylic acid group of bile salt and deeper penetration of bile acid molecules into TX-100 micelles at lower pH. Proposed molecular interactions are extremely informative to understand more about these biologically important compounds playing a crucial role in digestion processes.

The effect of hydroxyl moieties and their oxosubstitution on bile acid association studied in floating monolayers.

Bile salt aggregates are promising candidates for drug delivery vehicles due to their unique fat-solubilizing ability. However, the toxicity of bile salts increases with improving fat-solubilizing capability and so an optimal combination of efficient solubilization and low toxicity is necessary. To improve hydrophilicity (and decrease toxicity), we substituted hydroxyl groups of several natural bile acid (BA) molecules for oxogroups and studied their intrinsic molecular association behavior. Here we present the comparative Langmuir trough study of the two-dimensional (2D) association behavior of eight natural BAs and four oxoderivatives (traditionally called keto-derivatives) floated on an aqueous subphase. The series of BAs and derivatives showed systematic changes in the shape of the compression isotherms. Two types of association could be distinguished: the first transition was assigned to the formation of dimers through H-bonding and the second to the hydrophobic aggregation of BA dimers. Hydrophobic association of BA molecules in the films is linked to the ability of forming H-bonded dimers. Both H-bond formation and hydrophobic association weakened with increasing number of hydroxyl groups, decreasing distance between hydroxyl groups, and increasing oxosubstitution. The results also show that the Langmuir trough method is extremely useful in selecting appropriate BA molecules to design drug delivery systems.

Determination of number-average aggregation numbers of bile salts micelles with a special emphasis on their oxo derivatives—The effect of the steroid skeleton

BackgroundThe special geometry of the steroid skeleton causes that bile acid anions, in contrast to aliphatic amphiphiles, form micelles with a small aggregation number. MethodsThe number-average aggregation numbers (n¯) are determined using Moroi–Matsuoka–Sugioka thermodynamic method. Also, for analysed bile acid sodium salts functions between spin–lattice relaxation time (T1) and concentration of monomers (cBA−) are determined. ResultsFor 7-oxodeoxicholic (7-ODC) acid and hyodeoxicholic acid (HD) monomers, curve T1=fcBA− contains two inflexion points. Mentioned monomers and cholic acid anion (C) are influential observations in relation to a line of linear regression between n¯ and para\\meter of monomer hydrophobicity (lnk, retention capacity from RPHPLC). This suggests that, in micelles of bile acid anions: 7-ODC, HD and C, beside main, hydrophobic interactions, hydrogen bonds are also possible between building units. ConclusionThe increase in the number of oxo groups in the molecule is accompanied with a decrease in the hydrophobicity of the convex side of the steroid skeleton of the bile acid anion, resulting in a lower aggregation number. Obtained results indicate that C12 and C7 α-axial OH and oxo groups on the same C atoms of the investigated bile acid molecules have different spatial environment, which is confirmed by conformational analysis. General significanceDeviation from the linear model: number-average aggregation numbers with hydrophobicity of monomers, suggests the existence of additional, intermolecular interactions beside hydrophobic in micelles.

Between Peptides and Bile Acids: Self-Assembly of Phenylalanine Substituted Cholic Acids

Biocompatible molecules that undergo self-assembly are of high importance in biological and medical applications of nanoscience. Peptides and bile acids are among the most investigated due to their ability to self-organize into many different, often stimuli-sensitive, supramolecular structures. With the aim of preparing molecules mixing the aggregation properties of bile acid and amino acid-based molecules, we report on the synthesis and self-association behavior of two diastereomers obtained by substituting a hydroxyl group of cholic acid with a l-phenylalanine residue. The obtained molecules are amphoteric, and we demonstrate that they show a pH-dependent self-assembly. Both molecules aggregate in globular micelles at high pH, whereas they form tubular superstructures under acid conditions. Unusual narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm are formed by the derivatives. The diasteroisomer with α orientation of the substituent forms in addition a wider tubule (17 nm cross-section diameter). The ability to pack in supramolecular tubules is explained in terms of a wedge-shaped bola-form structure of the derivatives. Parallel or antiparallel face-to-face dimers are hypothesized as fundamental building blocks for the formation of the narrow and wide nanotubes, respectively.

Multi-component, Self-assembled, Functional Soft Materials

Supramolecular chemistry is an emerging tool for devising materials that can perform specified functions. The self-assembly of facially amphiphilic bile acid molecules has been extensively utilized for the development of functional soft materials. Supramolecular hydrogels derived from the bile acid backbone act as useful templates for the intercalation of multiple components. Based on this, synthesis of gel-nanoparticle hybrid materials, photoluminescent coating materials, development of a new enzyme assay technique, etc. were achieved in the author's laboratory. The present account highlights some of these achievements.

Molecular interactions between selected sodium salts of bile acids and morphine hydrochloride

The objective of this study was to understand the prolonged analgesic action of morphine hydrochloride observed in the presence of sodium 12-oxochenodeoxycholanate. Based on literature, this phenomenon may be due to the formation of aggregates in the cell between the molecules of bile acids and morphine. In addition to the sodium 12-oxochenodeoxycholanate, the present investigation also included salts of cholic and 7-oxodeoxycholic acids. Saturation transfer difference NMR experiments showed that morphine binds to the bile acid molecule close to the aromatic protons H1 and H2 provided that the concentration of the bile acid salt approaches the critical micellar concentration (CMC). The spin-lattice relaxation times (T1) of the affected protons decrease significantly in the presence of micellar solutions of the bile acid salts, and the most pronounced change in T1 was observed for sodium 7-oxodeoxycholate. Diffusion-ordered NMR experiments suggested that morphine hydrochloride can interact only with sodium 7-oxochenodeoxycholate. It can be supposed that the molecular ratio of sodium 7-oxodeoxycholate and morphine hydrochloride in the mixed micelle is 2:1. The CMC values of mixed micelles do not differ from the CMC values of the micelle constituents, which suggests that the binding of morphine hydrochloride does not perturb the hydrophobic domain of the bile acid molecule. In the presence of bile acids, the transfer rate constant (k12) of morphine hydrochloride from the buffered aqueous solution to chloroform (model of the cell membrane) shows a decrease. A significant decrease of the k12 was also observed in the presence of micellar solutions. Kinetic measurements indicated that, in addition to micellar interaction between morphine hydrochloride and sodium salts of bile acids, a complex may also be formed in chloroform via hydrogen bonds formed between the drug and bile acid molecules.

Effect of sodium salts of 3α,12α-dihydroxy-7-oxo-5β-cholanoic and 3,7,12-trioxo-5β-cholanoic acids on verapamil hydrochloride in biophysical-chemical model experiments

It is known that certain bile acids have a promotive effect on the action of some drugs. Special attention is paid to bile acids having oxo groups instead of OH groups in the steroid skeleton of their molecule, since these derivatives have a lower hemolytic potential (membrane toxicity). This study examined the effects of sodium salts of 3?,12?-dihydroxy-7-oxo-5?-cholanoic acid (7-oC) and 3,7,12-trioxo-5?- cholanoic acid (3,7,12-toC) on the adsorption of verapamil hydrochloride on activated carbon (model of the cell membrane). The interaction was followed by measuring the effect of verapamil on the functional dependence between the spin-lattice relaxation time T1 (protons of the C18 angular group of the bile acid molecule) and the bile acid concentration in deuterated chloroform (model of the cell membrane lipid phase). Whether a depot effect of verapamil exists when 7-oC and 3,7,12-toC (in the form of methyl esters) are present in chloroform was also investigated. It was found that 7-oC exhibited a significant effect in the experiments with verapamil, whereas 3,7,12-toC showed no difference of the measured parameters with respect to the control. This indicates that bile acid molecules should have OH groups bound to the steroid nucleus, in order to exhibit an effect on the monitored physico-chemical parameters of verapamil.

Effects of conjugated and unconjugated bile acids on the activity of theVibrio choleraeporin OmpT

During infection, the enteric pathogen Vibrio cholerae encounters a bile-containing environment. Previous studies have shown that bile and/or bile acids exert several effects on the virulence and physiology of the bacterial cells. These observations have led to the suggestion that bile acids may play a signaling role in infection. We have previously reported that the bile component deoxycholic acid blocks the general diffusion porin OmpT in a dose-dependent manner, presumably as it transits through the pore. V. cholerae colonizes the distal jejunum and ileum, where a mixture of various conjugated and unconjugated bile acids are found. In this work, we have used patch clamp electrophysiology to investigate the effects of six bile acids on OmpT. Two bile acids (deoxycholic and chenodeoxycholic acids) were found to block OmpT at physiological concentrations below 1 mM, while glycodeoxycholic acid was mildly effective and cholic, lithocholic and taurodeoxycholic acids were ineffective in this range. The block was also voltage-dependent. These observations suggest the presence of a specific binding site inside the OmpT pore. Since deconjugation is due to the activity of the endogenous flora, the preferential uptake of some unconjugated bile acids by OmpT may signal the presence of a hospitable environment. The results are also discussed in terms of the possible molecular interactions between the penetrating bile acid molecule and the channel wall.

QSPR study of the effect of steroidal hydroxy and oxo substituents on the critical micellar concentration of bile acids

Measurements of the fluorescence intensity of 1,6-diphenylhexatriene (DPH) as a probe molecule allowed the determination of critical micellar concentrations (CMCs) for 30 bile acid (BA) molecules belonging to three linear congeneric groups, with three (Group I) and two (Group II) oxygen atoms from OH or oxo groups bound to the steroid skeleton, and glyco conjugated cholic acid and glyco conjugated cholic acid derivatives (Group III). The CMC values are related to the structure of the steroid nucleus by constructing novel molecular descriptors with 2D and 3D characteristics of topological descriptors. Namely, with conventional topological descriptors (first-order connectivity index, third-order connectivity index, Wiener-index, Balaban-index, molecular topological index, cluster count, etc.) BA molecules from the same congeneric group have identical values, so that these descriptors cannot be used to form a new model for the given congeneric group. The linear regressions (models) obtained in this work for each congeneric group relate the CMCs to this new descriptor. Statistical parameters of these models, as well as their predictivity, indicate the significance of the obtained equations, that is that the micelle formation is influenced not only by the number of OH and oxo groups in the steroid nucleus but also by their steric environment.

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi-site ligand-protein binding and multi-step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D (1)H-(15)N line-shape analysis, which allows a reliable investigation of ligand binding occurring on micro- to millisecond timescales, have been extended to model a two-step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule-ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi-step events could be modelled, for several residues, with a two-step binding mechanism. The protein in the ligand-bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.