Microcalorimetric Titration Research Articles

Ubiquitous purine sensor modulates diverse signal transduction pathways in bacteria

Purines and their derivatives control intracellular energy homeostasis and nucleotide synthesis, and act as signaling molecules. Here, we combine structural and sequence information to define a purine-binding motif that is present in sensor domains of thousands of bacterial receptors that modulate motility, gene expression, metabolism, and second-messenger turnover. Microcalorimetric titrations of selected sensor domains validate their ability to specifically bind purine derivatives, and evolutionary analyses indicate that purine sensors share a common ancestor with amino-acid receptors. Furthermore, we provide experimental evidence of physiological relevance of purine sensing in a second-messenger signaling system that modulates c-di-GMP levels.

Exploring solute binding proteins in Pseudomonas aeruginosa that bind to γ-aminobutyrate and 5-aminovalerate and their role in activating sensor kinases.

The standard method of receptor activation involves the binding of signals or signal-loaded solute binding proteins (SBPs) to sensor domains. Many sensor histidine kinases (SHKs), which are activated by SBP binding, are encoded adjacent to their corresponding sbp gene. We examined three SBPs of Pseudomonas aeruginosa PAO1, encoded near the genes for the AgtS (PA0600) and AruS (PA4982) SHKs, to determine how common this arrangement is. Ligand screening and microcalorimetric studies revealed that the SBPs PA0602 and PA4985 preferentially bind to GABA (KD = 2.3 and 0.58 μM, respectively), followed by 5-aminovalerate (KD = 30 and 1.6 μM, respectively) and ethanoldiamine (KD = 2.3 and 0.58 μM, respectively). In contrast, AgtB (PA0604) exclusively recognizes 5-aminovaleric acid (KD = 2.9 μM). However, microcalorimetric titrations did not show any binding between the AgtS sensor domain and AgtB or PA0602, regardless of the presence of ligands. Similarly, bacterial two-hybrid assays did not demonstrate an interaction between PA4985 and the AruS sensor domain. Therefore, sbp and shk genes located nearby are not always functionally linked. We previously identified PA0222 as a GABA-specific SBP. The presence of three SBPs for GABA may be linked to GABA's role as a trigger for P. aeruginosa virulence.

Anion-Binding Properties of Short Linear Homopeptides.

A comprehensive thermodynamic and structural study of the complexation affinities of tetra (L1), penta (L2), and hexaphenylalanine (L3) linear peptides towards several inorganic anions in acetonitrile (MeCN) and N,N-dimethylformamide (DMF) was carried out. The influence of the chain length on the complexation thermodynamics and structural changes upon anion binding are particularly addressed here. The complexation processes were characterized by means of spectrofluorimetric, 1H NMR, microcalorimetric, and circular dichroism spectroscopy titrations. The results indicate that all three peptides formed complexes of 1:1 stoichiometry with chloride, bromide, hydrogen sulfate, dihydrogen phosphate (DHP), and nitrate anions in acetonitrile and DMF. In the case of hydrogen sulfate and DHP, anion complexes of higher stoichiometries were observed as well, namely those with 1:2 and 2:1 (peptide:anion) complexes. Anion-induced peptide backbone structural changes were studied by molecular dynamic simulations. The anions interacted with backbone amide protons and one of the N-terminal amine protons through hydrogen bonding. Due to the anion binding, the main chain of the studied peptides changed its conformation from elongated to quasi-cyclic in all 1:1 complexes. The accomplishment of such a conformation is especially important for cyclopeptide synthesis in the head-to-tail macrocyclization step, since it is most suitable for ring closure. In addition, the studied peptides can act as versatile ionophores, facilitating transmembrane anion transport.

Understanding the Interaction of Uranyl Cation with Two C-Pivot Tripodal Amides: Synthesis, Complexation, Microcalorimetry, and DFT Studies.

Complexation of uranyl ions with two structurally related C-pivotal tripodal amides with varying spacer lengths, synthesized for the first time, was studied by optical spectroscopy. In the tripodal amides, the coordination was through the carbonyl O atoms where the carbonyl groups were away from the central C-atom by three spacer atoms (LI) and four spacer atoms (LII), respectively. Increasing the spacer atoms going from LI to LII favors the complexation with the linear uranyl cations and results in stronger complex formation. The complexation heat between the uranyl cations and the two amide ligands was directly measured by microcalorimetric titrations. The complexation with both the ligands was driven by exothermic enthalpy and positive entropy changes. Formation of the complex proceeded by the replacement of water molecules from the primary coordination sphere of the uranyl cation. Both ligands formed bisolvated (ML2-type) complexes in which one unit of the ligand binds in a monodentate manner and the other in a bidentate mode. Density functional theory calculations further supported our experimental observations.

Inclusion complexes of nabumetone with β-cyclodextrins: Spectroscopic, spectrometric and calorimetric studies in solution

The complexation of nabumetone (NAB) with various cyclodextrins (CDs): β-cyclodextrin (β-CD), hydroxypropyl-β-CD (HPβCD), randomly methylated β-CD (RMβCD), and sulfobutylether sodium salt β-CD (SBEβCD), was investigated as a mean to increase the poor solubility of NAB both in water and simulated biorelevant media (pH = 1.0 (HCl) and pH = 4.5 and 6.8 (phosphate buffer)). An approximately 170 times increase in NAB solubility was achieved for SBEβCD in all media. The formation of 1:1 complexes was confirmed by phase-solubility measurements, spectrofluorimetric titration and high resolution mass spectrometry. Additionally, isothermal microcalorimetric titrations were employed to determine thermodynamic parameters (lgK, ΔrG0, ΔrH0, ΔrS0). Stability constants (lgK = 3.42; 3.41; 3.47 and 3.62 for NAB complexes with β-CD, HPβCD, RMβCD, and SBEβCD, respectively) agree with those obtained by phase-solubility experiments and spectrofluorimetric titrations. The inclusion was enthalpy and entropy favorable. NMR analyses showed two possible orientations of NAB in the β-CD cavity simultaneously exist in the solution, with a naphthalene ring inside the β-CD cavity and methoxy or butanone substituent oriented towards the narrow β-CD rim. All experimental results are in good agreement with those based on molecular modeling previously reported in the literature.

Amine-recognizing domain in diverse receptors from bacteria and archaea evolved from the universal amino acid sensor

Bacteria possess various receptors that sense different signals and transmit information to enable an optimal adaptation to the environment. A major limitation in microbiology is the lack of information on the signal molecules that activate receptors. Signals recognized by sensor domains are poorly reflected in overall sequence identity, and therefore, the identification of signals from the amino acid sequence of the sensor alone presents a challenge. Biogenic amines are of great physiological importance for microorganisms and humans. They serve as substrates for aerobic and anaerobic growth and play a role of neurotransmitters and osmoprotectants. Here, we report the identification of a sequence motif that is specific for amine-sensing sensor domains that belong to the Cache superfamily of the most abundant extracellular sensors in prokaryotes. We identified approximately 13,000 sensor histidine kinases, chemoreceptors, receptors involved in second messenger homeostasis and Ser/Thr phosphatases from 8,000 bacterial and archaeal species that contain the amine-recognizing motif. The screening of compound libraries and microcalorimetric titrations of selected sensor domains confirmed their ability to specifically bind biogenic amines. Mutants in the amine-binding motif or domains that contain a single mismatch in the binding motif had either no or a largely reduced affinity for amines. We demonstrate that the amine-recognizing domain originated from the universal amino acid-sensing Cache domain, thus providing insight into receptor evolution. Our approach enables precise "wet"-lab experiments to define the function of regulatory systems and therefore holds a strong promise to enable the identification of signals stimulating numerous receptors.

Thermochemical Analysis of Hydrogenation of Pd-Containing Composite Based on TiZrVNbTa High-Entropy Alloy

The microcalorimetric hydrogen titration technique combined with conventional volumetric measurements has been used to reveal peculiarities of the hydrogenation of the single-phase TiZrVNbTa equiatomic high-entropy alloy. The alloy has been produced in the form of microfibers by the pendent drop melt extraction technique. Palladium coating of the fibers has been applied to enable first hydrogenation at room temperature without additional activation. An analysis of the obtained data allows us to evaluate the dependence of hydrogenation enthalpy on the hydrogen concentration in the alloy. Three concentration ranges, presumably related to the formation of the hydrogen solid solution, monohydride and dihydride phases, have been identified, and the corresponding ΔH values of about −100, −80 and −60 kJ/mol H2, respectively, have been determined.

Understanding mucus modulation behavior of chitosan oligomers and dextran sulfate combining light scattering and calorimetric observations

This study reports the fundamental understanding of mucus-modulatory strategies combining charged biopolymers with distinct molecular weights and surface charges. Here, key biophysical evidence supports that low-molecular-weight (Mw) polycation chitosan oligosaccharides (COSs) and high-Mw polyanion dextran sulfate (DS) exhibit distinct thermodynamic signatures upon interaction with mucin (MUC), the main protein of mucus. While the COS → MUC microcalorimetric titrations released ~14 kcal/mol and ~60 kcal/mol, the DS → MUC titrations released ~1200 and ~1450 kcal/mol at pH of 4.5 and 6.8, respectively. The MPT-2 titrations of COS → MUC and DS → MUC indicated a greater zeta potential variation at pH = 4.5 (relative variation = 815 % and 351 %, respectively) than at pH = 6.8 (relative variation = 282 % and 136 %, respectively). Further, the resultant binary (COS-MUC) and ternary (COS-DS-MUC) complexes showed opposite behavior (aggregation and charge inversion events) according to the pH environment. Most importantly, the results indicate that electrostatics could not be the driving force that governs COS-MUC interactions. To account for this finding, we proposed a two-level abstraction model. Macro features emerge collectively from individual interactions occurring at the molecular level. Therefore, to understand the outcomes of mucus modulatory strategy based on charged biopolymers it is necessary to integrate both visions into the same picture.

Extraction and Complexation Investigation of Palladium(II) by a Nitrilotriacetate-Derived Triamide Ligand.

Effective and selective separation and recovery of the fission product palladium from high-level liquid waste are conducive not only to reducing its hazards to the public health and environment but also to alleviate the pressure on the increasing demand for natural palladium. Herein, the Pd2+ extraction in an HNO3 solution with a nitrilotriacetate-derived triamide ligand NTAamide(n-Oct) and the complexation between them were investigated. Using n-octanol as a diluent, NTAamide(n-Oct) demonstrated an excellent selectivity, strong extractability, and high loading capacity for Pd2+ extraction. Combined with the results of single-crystal X-ray diffraction, Fourier transform infrared spectroscopy, electrospray ionization-mass spectroscopy, microcalorimetric titration, and slope analysis, the extracted complexes were determined as [PdL2](NO3)2 and [PdL2][Pd(NO3)4] (where L denotes the NTAamide ligand) in 0.10 and 3.0 mol/L HNO3 solutions, respectively. The extraction model closely depended on the solvation state of Pd2+ in the HNO3 solution. An ion-pair extraction model was proposed and discussed.

Anion-Sensing Properties of Cyclopentaphenylalanine.

Cyclic pentaphenylalanine was studied as an efficient anion sensor for halides, thiocyanate and oxoanions in acetonitrile and methanol. Stability constants of the corresponding complexes were determined by means of fluorimetric, spectrophotometric, 1H NMR, and microcalorimetric titrations. A detailed structural overview of receptor–anion complexes was obtained by classical molecular dynamics (MD) simulations. The results of 1H NMR and MD studies indicated that the bound anions were coordinated by the amide groups of cyclopeptide, as expected. Circular dichroism (CD) titrations were also carried out in acetonitrile. To the best of our knowledge, this is the first example of the detection of anion binding by cyclopeptide using CD spectroscopy. The CD spectra were calculated from the structures obtained by MD simulations and were qualitatively in agreement with the experimental data. The stoichiometry of almost all complexes was 1:1 (receptor:anion), except for dihydrogen phosphate where the binding of dihydrogen phosphate dimer was observed in acetonitrile. The affinity of the cyclopeptide receptor was correlated with the structure of anion coordination sphere, as well as with the solvation properties of the examined solvents.

A comprehensive research on Lactone Sophorolipid (LSL) and Soy Protein Isolate (SPI) interacting mixture

Soy protein isolate (SPI) is a promising biological material, and lactone sophorolipid (LSL) is a non-toxic, completely biodegradable amphiphilic functional biosurfactant. A comprehensive research of interaction mode and mechanism on LSL and SPI composite system was studied using surface tension, turbidity, fluorescence spectroscopy, ultraviolet–visible (UV–Vis) spectroscopy, circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), isothermal microcalorimetric titration (ITC) and molecular docking methods. The properties of surface activity, foam stability, antibacterial ability and so on were evaluated in detail. Compared with a single pure substance, the performance of the composite system shows different performance optimizations in different aspects. For the SPI/LSL systems with different mass ratios studied, the surface activity, foaming performance and foam stability of the composite system with a mass ratio of 1:50 are more prominent than those of other ratios. The fluorescence distribution showed the increase of LSL leading to a significant change in the fluorescence distribution of SPI, the quenching mechanism is mainly static quenching mechanism, and the combination of LSL and SPI is an spontaneous endothermic process. UV–Vis spectroscopy showed that LSL changed the conformation of SPI. The CD spectrum indicated that the combination of LSL and SPI increases the α-helical structure of protein, and is beneficial to the secondary structure of protein. The molecular model presents the conformation of the SPI/LSL complex, indicating that the hydrophobic force and hydrogen bonds are considered to be the main interaction forces between SPI and LSL, which may lead to the flexible conformation of the protein. This research enriches our understanding of the interaction mechanism between SPI and LSL, and provides some important and interesting information for the development and application of biosurfactant-protein mixed systems with environmentally friendly, non-toxic and non-polluting properties.

2-Carboxamido-6-(1H-pyrazol-3-yl)-pyridines as ligands for efficient separation of americium(III) from europium(III)

The synthesis, Eu3+ complexation, and selective solvent extraction of Am3+ over Eu3+ in nitric acid solution by 2-carboxamido-6-(1H-pyrazol-3-yl)-pyridines (A-PzPy) ligands were described. Without the aid of any lipophilic anion source, A-PzPy ligands in methyl isobutyl ketone (MIBK) diluent demonstrated a more favorable affinity of Am3+ than Eu3+ at high acidity levels. Slope analysis showed the formation of 1:2 metal/ligand extracted species. The extraction was a spontaneous exothermic process. The analyses of fluorescence titration, Fourier transform infrared (FT-IR), Raman, time-resolved laser fluorescence spectrum (TRLFS), electrospray ionization mass spectrometry (ESI-MS), conductivity and microcalorimetric titration revealed that the composition of complex is [Eu(NO3)2(H2O)∙2(Et-p-Tol-A-PzPy)](NO3). Combining the results of solvent extraction and complexation study, the extraction mechanism was proposed. Furthermore, the stability constants (log β) and thermodynamic parameters (ΔH, ΔS, ΔG) for complexation of Eu3+ with Et-p-Tol-A-PzPy were also presented.

Thermodynamic and MD studies of anion complexation by cyclopentaleucine in acetonitrile and dimethyl sulfoxide

Complexation affinity of cyclic pentaleucine for halogen anions, thiocyanate and oxoanions was studied in acetonitrile and dimethyl sulfoxide. Equilibrium constants and derived Gibbs energies, as well as enthalpies and entropies of the corresponding reactions were determined by means of microcalorimetric and 1H NMR titrations. To get more detailed insight into the binding of anions by the cyclopeptide, classical molecular dynamics simulations were carried out. The preorganization of cyclopentaleucine binding site by inclusion of solvent molecules was observed in acetonitrile by these simulations. The investigated receptor showed larger affinity for all anions in acetonitrile than in dimethyl sulfoxide. The results of 1H NMR studies and those of molecular dynamics simulations indicated that the bound anions were coordinated by the amide groups of cyclopeptide. The stoichiometry of most of the complexes was 1:1 (receptor:anion), with the exception of dihydrogen phosphate and hydrogen sulfate complexes, where higher stoichiometries (1:2 and 2:1, respectively) were also observed. In the case of complexes with H2PO4– anion, in both acetonitrile and dimethyl sulfoxide the binding of dihydrogen phosphate dimer was observed, whereas the species in which two ligands coordinate one HSO4– ion was observed in acetonitrile. The obtained thermodynamic reaction parameters and the observed rather strong solvent effect on the studied equilibria were discussed regarding anions and receptor structural properties as well as solvation abilities of the solvents used.

The structural basis for signal promiscuity in a bacterial chemoreceptor.

Signalling through chemosensory pathways is typically initiated by the binding of signal molecules to the chemoreceptor ligand binding domain (LBD). The PcaY_PP chemoreceptor from Pseudomonasputida KT2440 is characterized by an unusually broad signal range, and minimal requisites for signal binding are the presence of a C6-membered ring and that of a carboxyl group. Previous studies have shown that only some of the multiple signals recognized by this chemoreceptor are of apparent metabolic value. We report here high-resolution structures of PcaY_PP-LBD in the absence and presence of four cognate chemoeffectors and glycerol. The domain formed a four-helix bundle (4HB), and both ligand binding sites of the dimer were occupied with the high-affinity ligands protocatechuate and quinate, whereas the lower-affinity ligands benzoate and salicylate were present in only one site. Ligand binding was verified by microcalorimetric titration of site-directed mutants revealing important roles of an arginine and number of polar residues that establish an extensive hydrogen bonding network with bound ligands. The comparison of the apo and holo structures did not provide evidence for this receptor employing a transmembrane signalling mechanism that involves piston-like shifts of the final helix. Instead, ligand binding caused rigid-body scissoring movements of both monomers of the dimer. Comparisons with the 4HB domains of the Tar and Tsr chemoreceptors revealed significant structural differences. Importantly, the ligand binding site in PcaY_PP-LBD is approximately 8Å removed from that of the Tar and Tsr receptors. Data indicate a significant amount of structural and functional diversity among 4HB domains. DATABASES: The coordinates and structure factors have been deposited in the protein data band with the following IDs: 6S1A (apo form), 6S18 (bound glycerol), 6S33 (bound protocatechuate), 6S38 (bound quinate), 6S3B (bound benzoate) and 6S37 (bound salicylate).

Highly Efficient Trivalent Americium/Europium Separation by Phenanthroline-Derived Bis(pyrazole) Ligands.

The synthesis, Eu3+ complexation, and solvent extraction of Am3+ and Eu3+ from nitric acid solutions by tetradentate phenanthroline-derived bis(pyrazole) (BPPhen) ligands were described. By using meta-nitrobenzotrifluoride as diluent, BPPhen ligands in combination with 2-bromohexanoic acid extracted Am3+ and Eu3+ with remarkably high efficiency, excellent selectivity, and fast extraction kinetics. Stripping posed no issues. The ligands also showed excellent hydrolytic stability and acid tolerance. 2-Bromohexanoic anion neutralized the charge and increased the lipophilicity of the extracted ion pair. The extraction conformed to a cation exchange model. Slope analysis demonstrated the extraction of 1:2 metal/ligand complexes. Analyses by electrospray ionization mass spectrometry, time-resolved laser-induced fluorescence spectroscopy, Raman, and Fourier transform infrared techniques indicated that the composition of the extracted species is [Eu(nOct-BPPhen)2(H2O)]3+. The formation of 1:2 complexes was also confirmed by UV-vis spectroscopic titration and microcalorimetric titration methods. Meanwhile, the stability constants ( K) and the thermodynamic parameters (Δ H, Δ S, Δ G) for the complexation of Eu3+ with nOct-BPPhen were presented too.

Complexation of Alkali Metal Cations by a Tertiary Amide Calix[4]Arene Derivative in Strongly Cation Solvating Solvents

The complexation of alkali-metal cations with calix[4]arene tertiary amide derivative (L) was studied in N- methylformamide (NMF), N, N- dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) by means of microcalorimetric and UV spectrophotometric titrations. The Gibbs energies, enthalpies, and entropies for transfer of reactants and products from N- methylformamide to other solvents were determined. Favorable enthalpic contribution to overall stability was found to be the most important for all complexation reactions, especially in the case of NaL+ formation, resulting in an affinity peak of L for this cation. The complexation entropy changes were always unfavorable. The ligand dissolution was endothermic in all solvents, accompanied by positive solution entropy. The highest complex stability constants were determined in NMF, whereas in DMSO the affinity of L towards alkali metal cations was the lowest. An interesting interplay between the transfer enthalpies and entropies of the reactants and complexes was revealed and discussed in detail.

Calorimetric study of peculiar hydrogenation behavior of nanocrystalline TiFe

Thermodynamic analysis of hydrogen interaction with TiFe nanocrystalline intermetallic compound produced by mechanical alloying has been carried out using microcalorimetric titration technique. The boundary of the phase regions and H2 absorption/desorption reaction enthalpies have been defined in comparison with conventional as-cast alloy. It has been concluded that specific microstructure and oxygen contamination determine the system features within the α solid solution region and play a key role in formation of β2 and γ, but does not affect the thermodynamic parameters of the main β → α transformation.

Rigid Organization of Fluorescence-Active Ligands by Artificial Macrocyclic Receptor to Achieve the Thioflavin T-Amyloid Fibril Level Association.

The push-pull molecules with an intramolecular charge transfer from donor to acceptor sides upon excitation exhibit a wide variety of biological and electronic activities, as exemplified by the in vivo fluorescence imaging probes for amyloid fibrils in the diagnosis and treatment of amyloid diseases. Interestingly, the structurally much simpler bis(4,8-disulfonato-1,5-naphtho)-32-crown-8 (DNC), in keen contrast to the conventional macrocyclic receptors, was found to dramatically enhance the fluorescence of twisted intramolecular charge-transfer molecules possessing various benzothiazolium and stilbazolium fluorophores upon complexation. Spectroscopic and microcalorimetric titrations jointly demonstrated the complex structures and the interactions that promote the extremely strong complexation, revealing that the binding affinity in these artificial host-guest pairs could reach up to a nearly 10(7) M(-1) order of magnitude in water, and the sandwich-type complexation is driven by electrostatic, hydrophobic, π-stacking, and hydrogen-bonding interactions. Quantum chemical calculations on free molecules and their DNC-bound species in both the ground and excited states elucidated that the encapsulation by DNC could greatly deter the central single and double chemical bonds from free intramolecular rotation in the singlet excited state, thus leading to the unique and unprecedented fluorescence enhancement upon sandwich-type complexation. This complexation-induced structural reorganization mechanism may also apply to the binding of other small-molecule ligands by functional receptors and contribute to the molecular-level understanding of the receptor-ligand interactions in many biology-related systems.

Solubility product and heat of formation of lead alkyl xanthates by microcalorimetric titration

Microcalorimetric titration of lead ions with various alkyl xanthate ions was used to determine the solubility product (Kso) and the heat of formation (ΔH0) of lead alkyl xanthates (PbX2). The titration was carried out at 25°C in a 25mL size isoperibol reactor containing lead aqueous solutions with an ionic strength of 0.01M and pH6. Sodium ethyl xanthate, potassium propyl xanthate, potassium butyl xanthate and potassium amyl xanthate were used in this study. A new relationship was found for the Kso and the number of carbon atoms (N) in the xanthate alkyl chain, namely logKso=−10.033−1.049N. The relationship between ΔH0 and N was found to be ΔH0=−62.29−7.218N, in kJ/mol PbX2. The Kso and ΔH0 values that were determined by microcalorimetry were compared to those theoretically calculated taking into account the transfer of the xanthate alkyl chain from the aqueous solution to the lead xanthate solid phase. There was an excellent agreement between theoretical and calorimetric ΔH0. Low deviation was found between theoretical and calculated Kso.

Preorganization of the catalytic Zn2 +-binding site in the HNH nuclease motif—A solution study

The structure of the active site in a metalloenzyme can be a key determinant of its metal ion binding affinity and catalytic activity. In this study, the conformational features of the Zn2+-binding HNH motif were investigated by CD-spectroscopy in combination with isothermal microcalorimetric titrations. Various point mutations, including T454A, K458A and W464A, were introduced into the N-terminal loop of the nuclease domain of colicin E7 (NColE7). We show that the folding of the proteins was severely disturbed by the mutation of the tryptophan residue. This points to the importance of W464, being a part of the hydrophobic core located close to the HNH-motif. ITC demonstrated that the Zn2+-binding of the mutants including the W464 site became weak, and according to CD-spectroscopic measurements the addition of the metal ion itself cannot fully recover the functional structure. Titrations with Zn2+-ion in the presence and absence of the Im7 protein proved that the structural changes in the unfolded mutant included the HNH-motif itself. The metal-binding of the NColE7 mutants could be, however, fully rescued by the complexation of Im7. This suggests that the formation of a preorganized metal-binding site – existing in the wild-type enzyme but not in the W464 mutants – was induced by Im7. The low nuclease activity of all W464A mutants, however, implies that the interactions of this tryptophan residue are required for precise location of the catalytic residues, i.e. for stabilization of the fine-structure and of the tertiary structure. Our results contribute to the understanding of the metal binding site preorganization.