Irina V. Terekhova

In vitro oligomerization of a membrane protein complex. liposome-based reconstitution of trimeric photosystem I from isolated monomers.

Many membrane proteins can be isolated in different oligomeric forms. Photosystem I (PSI), for example, exists in cyanobacteria either as a monomeric or as a trimeric complex. Neither the factors responsible for the specific trimerization process nor its biological role are known at present. In the filamentous cyanobacteriumSpirulina platensis, trimers in contrast to monomers show chlorophyll fluorescence emission at 760 nm. To investigate the oligomerization process as well as the nature of the long wavelength chlorophylls, we describe here an in vitro reconstitution procedure to assemble trimeric PS I from isolated purified PS I monomers. Monomers (and trimers) were extracted from S. platensis with n-dodecyl β-d-maltoside and further purified by perfusion chromatography steps. The isolated complexes had the same polypeptide composition as other cyanobacteria (PsaA–PsaF and PsaI–PsaM), as determined from high resolution gels and immunoblotting. They were incorporated into proteoliposomes, which had been prepared by the detergent absorption method, starting from a phosphatidylcholine:phosphatidic acid mixture solubilized by octylglucoside. After the addition of monomeric PS I (lipid:chlorophyll, 25:1), octylglucoside was gradually removed by the stepwise addition of Biobeads. The 77 K fluorescence emission spectrum of these proteoliposomes displays a long wavelength emission at 760 nm that is characteristic of PS I trimers, which indicates for the first time the successful in vitro reconstitution of PS I trimers. In addition, a high performance liquid chromatography analysis of complexes extracted from these proteoliposomes confirms the formation of structural trimers. We also could show with this system 1) that at least one of the stromal subunits PsaC, -D, and -E is necessary for trimer formation and 2) that the extreme long wavelength emitting chlorophyll is formed as a result of trimer formation.

Selective Na+/K+ Effects on the Formation of α-Cyclodextrin Complexes with Aromatic Carboxylic Acids: Competition for the Guest

We investigated the effects of K(+) and Na(+) ions on the formation of α-cyclodextrin complexes with ionized aromatic carboxylic acids. Using solution calorimetry and (1)H NMR, we performed the thermodynamic and structural investigation of α-cyclodextrin complex formation with benzoic and nicotinic acids in different aqueous solutions containing K(+) and Na(+) ions as well as in pure water. The experiments show that the addition of sodium ions to solution leads to a decrease in the binding constants of the carboxylic acids with α-cyclodextrin as compared to pure water and solutions containing potassium ions. From another side, the effect of potassium ions on the binding constants is insignificant as compared to pure water solution. We suggest that the selectivity of cation pairing with carboxylates is the origin of the difference between the effects of sodium and potassium ions on complex formation. The strong counterion pairing between the sodium cation and the carboxylate group shifts the equilibrium toward dissociation of the binding complexes. In turn, the weak counterion pairing between the potassium cation and the carboxylate group has no effect on the complex formation. We complemented the experiments with molecular modeling, which shows the molecular scale details of the formation of cation pairs with the carboxylate groups of the carboxylic acids. The fully atomistic molecular simulations show that sodium ions mainly form direct contact pairs with the carboxylate group. At the same time, potassium ions practically do not form direct contact pairs with the carboxylate groups and usually stay in the second solvation shell of carboxylate groups. That confirms our hypotheses that the selective formation of ion pairs is the main cause of the difference in the observed effects of sodium and potassium salts on the guest-host complex formation of α-cyclodextrin with aromatic carboxylic acids. We propose a molecular mechanism explaining the effects of salts, based on competition between the cations and α-cyclodextrin for binding with the ionized carboxylic acids.

Investigation of the pH-dependent complex formation between β-cyclodextrin and dipeptide enantiomers by capillary electrophoresis and calorimetry

The effect of pH on complex formation between beta-CD and the enantiomers of the dipeptides Ala-Phe, Ala-Tyr and Asp-PheOMe was investigated at 298.15 K by CE and calorimetry. Beta-CD displayed a higher enantioselectivity toward the protonated peptides compared to their zwitterionic forms. While stronger binding of the DD-enantiomers than the LL-stereoisomers were found by calorimetry regardless of the ionization state of the peptides, essentially equal complexation constants of the enantiomers were determined by CE for the zwitterionic species of the peptides. The reversal of the enantiomer migration order observed in CE was attributed primarily to a stereoselective complexation-induced pK(a) shift. In calorimetry, complexation of the protonated DD-enantiomers by beta-CD was accompanied by higher enthalpy and entropy changes resulting in more stable complexes compared to the LL-peptides. The enthalpy and entropy of complexation was affected by pH and peptide structure.

Thermodynamics of the interactions of peptides with α- and β-cyclodextrins

The enthalpies of solution of α- and β-cyclodextrins is aqueous peptide solutions were determined experimentally at 298.15 K. The obtained results were used to calculate pair cross interaction parameters between solutes. The results are discussed in terms of the likelysolute–solute interactions. For systems α-cyclodextrin+peptide and β-cyclodextrin+peptide the diametrically opposite character of interaction defined by structure and solvation of the molecules is observed.

Molecular recognition of aromatic carboxylic acids by hydroxypropyl-γ-cyclodextrin: experimental and theoretical evidence

Inclusion complex formation of hydroxypropyl-γ-cyclodextrin with benzoic, nicotinic and isomeric aminobenzoic acids in water was studied by calorimetry, 1H NMR, densimetry and molecular modeling. It was observed that hydroxypropyl-γ-cyclodextrin selectively interacts with the considered acids forming stable inclusion complexes of 1 : 2 stoichiometry with benzoic and p-aminobenzoic acids, which exist in aqueous solution predominantly as neutral molecules. The binding affinity of hydroxypropyl-γ-cyclodextrin to m-aminobenzoic and nicotinic acids having the zwitterionic structure is considerably lower and a 1 : 1 inclusion complex is formed only with the former. The binding mode and thermodynamic parameters of complex formation were evaluated. It was shown that they strongly depend on the structure and ionization state of the acids. An efficient molecular modeling approach for simulating the encapsulation process for 1 : 1 and 1 : 2 stoichiometries was developed and implemented. A good agreement between experimental and theoretical results was demonstrated.

α-Cyclodextrin/aminobenzoic acid binding in salt solutions at different pH: dependence on guest structure.

Influence of Na(+) and K(+) cations on α-cyclodextrin guest-host complex formation with isomeric aminobenzoic acids was examined at different pH and temperature of 298.15 K by (1)H NMR and calorimetry methods. More pronounced influence of Na(+) on inclusion complex formation of α-CD with aminobenzoic acid anions compare to the effects of Na(+) on α-CD complex formation with zwitterionic aminobenzoic acid molecules was revealed. For the first time, the dependence of salt effects on the structure, ionization and the hydration state of the guest molecule was demonstrated and analysed on the basis of the obtained thermodynamic parameters of complex formation and calculated free energy of hydration of different ionized forms of aminobenzoic acids.

Thermodynamic characteristics of the formation of α- and β-cyclodextrin complexes with lumichrome, lumazine, and uracil in aqueous solution

Interactions of α- and β-cyclodextrins with lumichrome and its structural fragments, lumazine and uracil, were studied by means of solubility and 1H NMR spectroscopy. α-Cyclodextrin was found to have a weak complexing ability toward the studied compounds. It was established that β-cyclodextrin forms stable complexes with lumichrome and does not complex with lumazine and uracil. It was shown that only the benzene ring of lumichrome penetrates the β-cyclodextrin cavity, leading to a substantial increase in the solubility of lumichrome in water. We concluded that β-cyclodextrin complexation with lumichrome is highly exothermic due to the van der Waals interactions and hydrogen bonding between polar groups of the reagents.

Cocrystal formation, crystal structure, solubility and permeability studies for novel 1,2,4-thiadiazole derivative as a potent neuroprotector

&NA; The cocrystallization approach has been applied to modify the poor solubility profile of the biologically active 1,2,4‐thiadiazole derivative (TDZ). Extensive cocrystal screening with a library of coformers resulted in formation of a new solid form of TDZ with vanillic acid in a 1:1 molar ratio. The cocrystalline phase was identified and characterized by thermal and diffraction analyses including single‐crystal X‐ray diffraction. The energies of intermolecular interactions in the crystal were calculated by solid‐state DFT and PIXEL methods. Both calculation schemes show good consistency in terms of total energy of the intermolecular interactions and suggest that the cocrystal is mainly stabilized via hydrogen bonds, which provide ca. 44% of the lattice energy. Since the cocrystal contained the hydroxybenzoic acid derivative as a coformer, the solubility profile of the cocrystal was investigated at different pHs using eutectic concentrations of the components. Furthermore, the influence of the cocrystallization on the permeability performance of the 1,2,4‐thiadiazole through an artificial regenerated cellulose membrane was also evaluated. In addition, the thermodynamic functions of the cocrystal formation were estimated from the solubility of the cocrystal and the corresponding solubility of the pure compounds at various temperatures. The cocrystal formation process was found to have a relatively small value of the driving force (−5.3 kJ·mol−1). The most significant contribution to the Gibbs energy was provided by the exothermic enthalpy of formation. Graphical abstract Figure. No caption available.

Cyclodextrin–benzoic acid binding in salt solutions: Effects of biologically relevant anions

Inclusion complex formation of benzoic acid with α-, β- and γ-cyclodextrins in water and in 0.2 M solutions of inorganic salts (KCl, KBr, KH2PO4 and K2SO4) has been studied by means of 1H NMR at 298.15 K. Binding constants have been determined and role of biologically active inorganic anions in the inclusion complex formation has been revealed. It has been shown that effects of the anions are determined not only by changing the ionic strength. More pronounced influence of Br- and H2PO4- compared with Cl- and SO4(2-) is caused by specific ion-molecular interactions, occurrence of which depends on the physical-chemical properties of the anions as well as on the binding mode of cyclodextrins with benzoic acid. Competing interactions of cyclodextrin-anion were observed in the presence of KBr, while the ternary complex formation was detected upon addition of KH2PO4.

New water-soluble dosage forms of 1,2,4-thiadiazole derivative on the basis of inclusion complexes with cyclodextrins

Effect of cyclodextrins on aqueous solubility of 1-[5-(3-chloro-phenylamino)-1,2,4-thiadiazol-3-yl]-propan-2-ol (I), which was synthesized and proposed for the treatment of Alzheimer’s disease, was studied. First of all, inclusion complex formation of I with different cyclodextrins was studied in aqueous solutions. Formation of more stable complexes with β- and hydroxypropyl-β-cyclodextrins was revealed. Next, solid inclusion complexes of I with β- and hydroxypropyl-β-cyclodextrins were prepared by the mechanical grinding, and their existence in the solid state was confirmed by powder X-ray diffraction, FTIR spectroscopy, microscopy, and thermochemical methods. Dissolution testing of the tablets of pure I and its complexes with β- and hydroxypropyl-β-cyclodextrins in the aqueous buffered solutions simulated the biological environment was carried out. The observed drastic enhancement of dissolution extent and dissolution rate of the complexes was attributed to the inclusion complex formation.