Orsolya Toke

Chiral separation by a monofunctionalized cyclodextrin derivative: From selector to permethyl-β-cyclodextrin bonded stationary phase

Preparation of (6-monoureido-6-monodeoxy) permethylated beta-cyclodextrin bonded chiral stationary phase from permethylated 6-monoamino-6-monodeoxy-beta-cyclodextrin is described. The optimized chiral stationary phase was evaluated by using HPLC separation of racemates of coumarin derivatives. Column characterization was performed by solid-state (13)C, (15)N, (29)Si NMR using cross-polarization at the magic angle spinning. The development process was supported by CE experiments where the complex formation between cyclodextrins and warfarin was investigated. The results demonstrate good enantio-discrimination for coumarin derivatives.

Internal motions and exchange processes in human ileal bile acid binding protein as studied by backbone 15N nuclear magnetic resonance spectroscopy

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone (15)N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the (15)N-(1)H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C-D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ β-strands and the C-D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins.

Molecular structure of bismuth trichloride from combined electron diffraction and vibrational spectroscopic study

The results of an electron diffraction reanalysis, augmented with a combined electron diffraction and vibrational spectroscopic elucidation, of the molecular structure of BiCl3 are reported. The principal parameters arerg(Bi-Cl)=2.424±0.005 å (rα=2.417±0.005 å) and <αCl-Bi-Cl=97.5±0.2‡. They are in excellent agreement with previous electron diffraction analysis [1], utilizing a more limited data range from the same experiment. They are also fully consistent with the expected trends of geometrical variation in the Group V trihalide series. The force fields of BiCl3, determined by normal coordinate analysis and by combined analysis, agree within experimental error.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR.

Besides aiding digestion, bile salts are important signal molecules exhibiting a regulatory role in metabolic processes. Human ileal bile acid binding protein (I‐BABP) is an intracellular carrier of bile salts in the epithelial cells of the distal small intestine and has a key role in the enterohepatic circulation of bile salts. Positive binding cooperativity combined with site selectivity of glycocholate and glycochenodeoxycholate, the two most abundant bile salts in the human body, make human I‐BABP a unique member of the family of intracellular lipid binding proteins. Solution NMR structure of the ternary complex of human I‐BABP with glycocholate and glycochenodeoxycholate reveals an extensive network of hydrogen bonds and hydrophobic interactions stabilizing the bound bile salts. Conformational changes accompanying bile salt binding affects four major regions in the protein including the C/D, E/F and G/H loops as well as the helical segment. Most of these protein regions coincide with a previously described network of millisecond time scale fluctuations in the apo protein, a motion absent in the bound state. Comparison of the heterotypic doubly ligated complex with the unligated form provides further evidence of a conformation selection mechanism of ligand entry. Structural and dynamic aspects of human I‐BABP–bile salt interaction are discussed and compared with characteristics of ligand binding in other members of the intracellular lipid binding protein family.

Temperature dependence of backbone dynamics in human ileal bile acid-binding protein: Implications for the mechanism of ligand binding

Human ileal bile acid-binding protein (I-BABP), a member of the family of intracellular lipid binding proteins plays a key role in the cellular trafficking and metabolic regulation of bile salts. The protein has two internal and, according to a recent study, an additional superficial binding site and binds di- and trihydroxy bile salts with positive cooperativity and a high degree of site-selectivity. Previously, in the apo form, we have identified an extensive network of conformational fluctuations on the millisecond time scale, which cease upon ligation. Additionally, ligand binding at room temperature was found to be accompanied by a slight rigidification of picosecond-nanosecond (ps-ns) backbone flexibility. In the current study, temperature-dependent (15)N NMR spin relaxation measurements were used to gain more insight into the role of dynamics in human I-BABP-bile salt recognition. According to our analysis, residues sensing a conformational exchange in the apo state can be grouped into two clusters with slightly different exchange rates. The entropy-enthalpy compensation observed for both clusters suggests a disorder-order transition between a ground and a sparsely populated higher energy state in the absence of ligands. Analysis of the faster, ps-ns motion of (15)N-(1)H bond vectors indicates an unusual nonlinear temperature-dependence for both ligation states. Intriguingly, while bile salt binding results in a more uniform response to temperature change throughout the protein, the temperature derivative of the generalized order parameter shows different responses to temperature increase for the two forms of the protein in the investigated temperature range. Analysis of both slow and fast motions in human I-BABP indicates largely different energy landscapes for the apo and holo states suggesting that optimization of binding interactions might be achieved by altering the dynamic behavior of specific segments in the protein.

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy

Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca2+ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α‐helical conformation in 50% CD3OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca2+ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain. Copyright

Structural ensembles based on NMR parameters suggest a complex pathway of ligand binding in human gastrotropin

Gastrotropin, the intracellular carrier of bile salts in the small intestine, binds two ligand molecules simultaneously in its internal cavity. The molecular rearrangements required for ligand entry are not yet fully clear. To improve our understanding of the binding process we combined molecular dynamics simulations with available structural and dynamic NMR parameters. The resulting ensembles reveal two distinct modes of barrel opening with one corresponding to the transition between the apo and holo states, whereas the other affecting different protein regions in both ligation states. Comparison of the calculated structures with NMR-derived parameters reporting on slow conformational exchange processes suggests that the protein undergoes partial unfolding along a path related to the second mode of the identified barrel opening motion.

Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid‐binding protein

Human ileal bile acid‐binding protein (I‐BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid‐binding proteins (iLBPs), disorder–order transitions and local unfolding processes are thought to mediate ligand entry and release in human I‐BABP. To gain insight into the stability of various protein regions, the temperature response of human I‐BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue‐specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate β‐strands contains a substantial amount of residual structure, whereas several segments of the C‐terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the β‐barrel, the E–F region and part of the helical cap. MD simulations up to 1 μs show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D–E turn and E–F region in the initiation of unfolding. The response of human I‐BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.