Molecular Simulation of Adsorption of Guest Molecules in Zeolitic Materials: A Comparative Study of Intermolecular Potentials

Abstract

Grand Canonical Monte Carlo simulation, together with an appropriate guest-host forcefield is shown to provide reasonably accurate predictions of adsorption properties of guest molecules in a variety of zeolitic materials. The use of a simple guest-host Kiselev-type potential permits the calculations to capture the essence of the behavior of simple guest-host systems such as rare gases or methane molecules in neutral AlPO₄-5. However, a full scale potential is needed in the more complex cases of large anisotropic molecules adsorbed in cationic zeolites (such as xylene isomers in faujasite). The guest-host potential model developed by Nicholson and coworkers is shown to allow an excellent transferability of the potential parameters from one guest/host system to another.     

Vitamin B<Subscript>12</Subscript> as a carrier for targeted platinum delivery: in vitro cytotoxicity and mechanistic studies

It is attractive to use vitamin B₁₂ as a carrier for targeted delivery of cytotoxic agents such as platinum complexes owing to the high demand for vitamin B₁₂ by fast proliferating cells. The basic {B₁₂–CN–Pt^II} conjugates are recognized by intracellular enzymes and converted to coenzyme B₁₂ in an enzymatic adenosylation assay. The reductive adenosylation of {B₁₂–CN–Pt^II} conjugates leads to the release of the Pt^II complexes; thus, {B₁₂–CN–Pt^II} conjugates can be considered as prodrugs. It is important not only to elucidate the activity of the cisplatin–B₁₂ conjugates, but also to understand the mode of action on a molecular level. Chemical reduction of {B₁₂–CN–Pt^II} conjugates with cobaltocene yielded cob(II)alamin and induced release of the corresponding Pt^II species. Kurnakov tests and coordination of 2′-deoxyguanosine or GMP to the released Pt^II complexes allowed isolation and characterization of Pt^II complexes as released during enzymatic adenosylation. The biological activity of these Pt^II complexes was evaluated. Since the cleaved Pt^II complexes show cytotoxicity, the {B₁₂–CN–Pt^II} conjugates can be used for specific targeting of cancer cells and therapeutic drug delivery. Preliminary in vitro cytotoxicity studies indicated lower activity (IC₅₀ between 8 and 88 μM) than found for pure cisplatin. Since active transport and receptor-mediated uptake limits the intracellular {B₁₂–CN–Pt^II} concentration, comparison with pure cisplatin is of limited use. We could show that the Pt^II complexes cleaved from B₁₂ exerted a cytotoxicity comparable to that of cisplatin itself. Cytotoxicity studies in vitamin B₁₂ free media showed a dependence on the addition of transcobalamin II for B₁₂–Pt(II) conjugates.     

Development and in vitro evaluation of an oral floating matrix tablet formulation of diltiazem hydrochloride

The purpose of this research was to prepare a floating drug delivery system of diltiazem hydrochloride (DTZ). Floating matrix tablets of DTZ were developed to prolong gastric residence time and increase its bioavailability. Rapid gastrointestinal transit could result in incomplete drug release from the drug delivery system above the absorption zone leading to diminished efficacy of the administered dose. The tablets were prepared by direct compression technique, using polymers such as hydroxypropylmethylcellulose (HPMC, Methocel K100M CR), Compritol 888 ATO, alone or in combination and other standard excipients. Sodium bicarbonate was incorporated as a gas-generating agent. The effects of sodium bicarbonate and succinic acid on drug release profile and floating properties were investigated. A 3² factorial design was applied to systematically optimize the drug release profile. The amounts of Methocel K100M CR (X₁) and Compritol 888 ATO (X₂) were selected as independent variables. The time required for 50% (t₅₀) and 85% (t₈₅) drug dissolution were selected as dependent variables. The results of factorial design indicated that a high level of both Methocel K100M CR (X₁) and Compritol 888 ATO (X₂) favors the preparation of floating controlled release of DTZ tablets. Comparable release profiles between the commercial product and the designed system were obtained. The linear regression analysis and model fitting showed that all these formulations followed Korsmeyer and Peppas model, which had a higher value of correlation coefficient (r). While tablet hardness had little or no effect on the release kinetics and was found to be a determining factor with regards to the buoyancy of the tablets. Published: September 7, 2007     

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A3

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co^II(Chro)₂ complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6 mM concentration of spermine completely abolished the DNA-binding activity of Fe^II(Chro)₂ and Cu^II(Chro)₂ and interfered with the associative binding of Co^II(Chro)₂ complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2 mM) promoted DNA strand cleavage by Cu^II(Chro)₂, whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co^II(Chro)₂ or Fe^II(Chro)₂. Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe^II(Chro)₂. Despite the fact that Cu^II(Chro)₂ and Fe^II(Chro)₂ demonstrated lower DNA-binding activity than Co^II(Chro)₂ in the absence of spermine, while Cu^II(Chro)₂ and Fe^II(Chro)₂ exhibited greater cytoxicity against HepG2 cells than Co^II(Chro)₂, possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.     

Modelling of the mechanical and mass transport properties of auxetic molecular sieves: an idealised organic (polymeric honeycomb) host–guest system

Force field-based simulations have been employed to model the mechanical properties of a range of undeformed molecular polymeric honeycombs having conventional and re-entrant hexagon pores. The conventional and re-entrant hexagon honeycombs are predicted to display positive and negative in-plane Poisson's ratios, respectively, confirming previous simulations. The structure, and mechanical and mass transport properties of a layered re-entrant honeycomb ((2,8)-reflexyne) were studied in detail for a uniaxial load applied along the x ₂ direction. The mechanical properties are predicted to be stress- (strain-) dependent and the trends can be interpreted using analytical expressions from honeycomb theory. Transformation from negative to positive Poisson's ratio behaviour is predicted at an applied stress of σ₂ = 2 GPa. Simulations of the loading of C₆₀ and C₇₀ guest molecules into the deformed layered (2,8)-reflexyne host framework demonstrate the potential for tunable size selectivity within the host framework. The entrapment and release of guest molecules is attributed to changes in the size and shape of the pores in this host–guest system.     

Thermal effects in guest–host systems: [Zn2(bdc)(S‐lac)(dmf)]•PhEtOH: A DSC and NMR study

Differential scanning calorimetry and nuclear magnetic resonance were used to investigate thermal effects in the guest–host systems where homochiral metal–organic sorbent [Zn₂(bdc)(S ‐lac)(dmf)] is considered as a host while 1‐phenylethanol enantiomers and their racemic mixture serve as guest molecules. A maximum energy gain from the guest–host interaction was observed in the system with the racemic mixture. The effect of host–guest recognition was revealed for the case of the host and guest having a similar type of chirality in the presence of antipode guest molecules.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 °C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [³¹P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H_II) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Topography of cyclodextrin inclusion complexes : Part I. Classification of crystallographic data of α-cyclodextrin inclusion complexes

The crystallographic and stoichiometric data obtained for 17 different inclusion complexes of α-cyclodextrin are reported. The cell dimensions and space-group symmetries reflect the packing arrangement of the torus-shaped host molecules and are largely determined by the size and ionic character of the guest molecules.In the series acetic acid, propionic acid, butyric acid, valeric acid, the first three complexes with α-cyclodextrin crystallize in a cage-type structure with space group P2₁2₁2₁, which is characteristic or small, non-ionic guest molecules. The valeric acid molecule seems to be too long to be accommodated in a cage structure; thus, the α-cyclodextrin molecules are arranged such that a structure consisting of parallel channels is formed. This packing is typical for the inclusion of long, thin, or ionic guest molecules. A third class of complexes with structures differing from the two described was also observed.A correlation exists between the type of inclusion complex and the volume required for a complex molecule: 1200–≈ 1400 ų for molecular guests, and 1400–1500 ų for ionic guests.     

Hydroxymethylnitrofurazone:Dimethyl-β-cyclodextrin Inclusion Complex: A Physical–Chemistry Characterization

Hydroxymethylnitrofurazone (NFOH) is active against Trypanosoma cruzi; however, its low solubility and high toxicity precludes its current use in treatment of parasitosis. Cyclodextrin can be used as a drug carrier system, as it is able to form inclusion (host–guest) complexes with a wide variety of organic (guest) molecules. Several reports have shown the interesting use of modified β-cyclodextrins in pharmaceutical formulation, to improve the bioavailability of drugs and to decrease their toxicity. The aim of this work was to characterize inclusion complexes formed between NFOH and dimethyl-β-cyclodextrin (DM-β-CD) by complexation/release kinetics and solubility isotherm experiments using ultraviolet (UV)-visible spectrophotometry and by the measurement of the dynamics information obtained from T ₁ relaxation times and diffusion (DOSY) experiments using nuclear magnetic resonance (NMR) spectroscopy. The complex was prepared at different NFOH and DM-β-CD molar ratios. The UV-visible measurements were recorded in a spectrophotometer, and NMR experiments were recorded at 20°C on a NMR spectrometer (Varian Inova) operating at 500 MHz. Longitudinal relaxation times were obtained by the conventional inversion-recovery method and the DOSY experiments were carried out using the BPPSTE sequence. The kinetics of complexation revealed that 30 h is enough for stabilization of the NFOH absorbance in presence of cyclodextrin. Solubility isotherm studies show a favorable complexation and increase in solubility when NFOH interacts with cyclodextrin. The analysis of the NMR-derived diffusion coefficients and T ₁ relaxation times shows that in the presence of DM-β-CD, NFOH decreases its mobility in solution, indicating that this antichagasic compound interacts with the cyclodextrin cavity. The release kinetics assays showed that NFOH changes its release profile when in the presence of cyclodextrin due to complexation. This study was focused on the physicochemical characterization of drug-delivery formulations that may serve as potentially new therapeutic options for the treatment of Chagas’ disease.     

Modulation and Optimization of Drug Release from Uncoated Low Density Porous Carrier Based Delivery System

The purpose of this research work was to explore an application of uncoated porous drug carrier prepared by single-step drug adsorption for a delivery system based on integration of floating and pulsatile principles intended for chronotherapy. This objective was achieved by utilizing 3² factorial design, solvent volume (X ₁) and drug amount (X ₂) as selected variables, for drug adsorption using solvents, methanol, and dichloromethane (DCM), of varying polarity. Nitrogen adsorption (N₂), scanning electron microscopy of cross-sections, and atomic force microscopy were done to study adsorption patterns and their effect on release pattern. Drug release study was customized by performing for 6 h in acidic environment to mimic gastroretention followed by basic environment akin to transit phase. Correlation between porous data from mercury and N₂ adsorption was probably studied for the first time. Observed regression analysis values for pore volume, surface area, and drug release indicated the influence of selected variables. Total release range in acidic medium was 12.77–24.57% for methanol, 8.79–15.26% for DCM, and final release of 69.45–92.23% for methanol, and 60.16–99.99% for DCM influenced by varying internal geometries was observed. Present form of drug delivery system devoid of any additives/excipients influencing drug release shows distinct behavior from other approaches/technologies in chronotherapy by (a) observing desired low drug release (8%) in acidic medium, (b) overcoming the limitations of process variables caused by multiple formulation steps and different characteristic polymers, (c) reducing time consumption due to single step process, and (d) extending as controlled/extended release.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Molecular simulation of cage occupancy and selectivity of binary THF–H2 sII hydrate

The hydrogen capacity of the binary THF–H₂ sII hydrate is determined by the cage occupancy and by the selectivity of guest molecules. Grand canonical Monte Carlo (GCMC) simulation is used to study the cage occupancy and selectivity of guest molecules from the equilibrium configuration of the binary sII hydrate. The cage framework is regarded as a rigid body and the number of guest molecules is varied to preserve the grand canonical ensemble. The occupancy and selectivity were investigated at a temperature of 270 K for pressures ranging from 0.1 to 200 MPa. It was found that most large cages select THF as guest molecules while small cages include only hydrogen molecules. Multiple occupancy of hydrogen, up to four molecules in large cages and two molecules in small cages, was found as the pressure increases. GCMC results show that the hydrogen capacity is approximately 1.1 wt% at 200 MPa.     

Oleoylethanolamide-Based Lyotropic Liquid Crystals as Vehicles for Delivery of Amino Acids in Aqueous Environment

We have investigated the phase behavior of self-assembled lyotropic liquid crystals (LC) formed by ternary mixtures of oleoylethanolamide (OEA), water and arginine. OEA, a natural analog of the endogenous cannabinoid anandamide involved in the peripheral regulation of feeding, was selected as a main component due to its capacity to induce efficient decreases in food intake and gains in body mass. Arginine was selected as representative hydrophilic amino acid and added to the OEA-water mixture at different concentrations. The phase diagrams were determined by combining cross-polarized optical microscopy and small angle x-ray scattering. First, the phase diagram for the OEA-water system was determined. It was shown that these two compounds give rise to reverse Ia3d double gyroid and reverse Pn3m double diamond cubic phases existing in bulk over a large window of temperature and composition, and that for water content beyond 25% Pn3m coexisted with excess water. Successively, the influence of arginine as guest molecule in the water channels of the reverse LC was investigated. For the sake of comparison, results for the OEA-water-arginine system were compared with analog series of OEA-water-glucose. The results showed that, at a fixed water content and temperature, the phase behavior of the liquid crystalline phases is strongly dependent on arginine concentration. In more detail, arginine could be encapsulated in the bulk OEA-water LC up to 2.0% wt, whereas transitions from Ia3d to Pn3m cubic phase were observed with increasing arginine concentration. Interestingly, upon an increase of water concentration beyond 20-25%, Pn3m phase started to coexist with excess water releasing the arginine in external water solution. Quantitative measurements of arginine content inside the LC water channels and in the excess external water solution revealed a complete release of the amino acid, demonstrating that the investigated lyotropic liquid crystalline systems can be used as ideal vehicles for the delivery of functional hydrophilic active molecules in aqueous environment.     

The effect of storage and active ingredient properties on the drug release profile of poly(ethylene oxide) matrix tablets

Different molecular weight forms of poly(ethylene oxide) can be used successfully in controlled release drug delivery due to their excellent matrix forming properties. Drug release of these materials follows nearly zero order kinetics, and is mainly governed by polymer swelling and erosion and diffusion of drug molecules. Because of its partly amorphous structure, poly(ethylene oxide) undergoes structural changes caused by elevated temperature and relative humidity of the storage medium resulting in an increased drug release. This physical process can be highly influenced by the structure of different drug molecules, such as polymer-binding ability and hydration tendency. These properties of two basic drugs embedded into poly(ethylene oxide) matrices were characterized by molecular modelling and an attempt was made to reveal their effect on the change of drug release stability, a prerequisite of the marketing authorization of dosage forms. The findings suggest that both the hydration properties of the active ingredient and the molecular weight of the polymer influence the effect of physical ageing of poly(ethylene oxide) on the drug release properties of the matrix.     

Quantum efficiency and antenna size of Photosystems IIα, IIβ and I in tobacco chloroplasts

Reaction center concentrations were determined in chloroplasts of tobacco, cv John William's Broadleaf, and its mutants Su/su and Su/su var. Aurea. Quantum yields of the primary reactions of Photosystems I, II_α and II_β (Melis, A. and Homann, P.H. (1975) Photochem. Photobiol. 21, 431–437) were obtained by measurement of their rate constants and the absorbed energy, under conditions where all three photosystems operated simultaneously and produced almost irreversibly a single charge separation.The concentrations and reaction rates of the photosystems were different in chloroplasts from the wild type and the mutants, but in chloroplasts of each type of plant used essentially all quanta absorbed by chlorophyll caused a charge separation in PS I, PS II_α or PS II_β. Since the quantum efficiency of each photosystem was close to one, kinetic differences between the photosystems and between different kinds of chloroplasts were only due to differences in antenna size. From the rate constants the number of chlorophyll molecules in the antenna of each photosystem could be calculated. It is argued that PS II_α and PS II_β must be different, independent structures.     

WIND-PVPA: Water/Ion NMR Detected PVPA to assess lipid barrier integrity in vitro through quantification of passive water- and ion transport

Water/Ion NMR Detected – Phospholipid Vesicle Permeability Assay (WIND-PVPA), is presented as a novel, straightforward and automatable method to assess lipid barrier integrity in vitro. The apparent permeability constants of water- and ions across the PVPA barriers are determined in a one-pot experiment under the influence of membrane-active guest molecules. NMR spectroscopy is used to quantify the water directly (D₂O) and the ions indirectly (complexed with EDTA) as a function of time. WIND-PVPA is demonstrated using four anti-microbial peptides, to show that membrane active molecules can be differentiated by their disruptive influence on the PVPA system. The results obtained are compared with explicit molecular dynamics simulations of lipid bilayers, AMPs, water and salt, where the motions of all individual water molecules relative to the lipid bilayer are monitored over the course of the simulations, allowing the calculation of theoretical apparent permeability constants of the corresponding single bilayer systems.Proof-of-principle is presented that WIND-PVPA can be used to evaluate the lipid barrier destabilizing effect of active guest molecules by measuring changes in passive water- and ion permeabilities upon exposure. The method is highly flexible in terms of barrier composition, choice of probes and membrane active compounds.     

Bilayer structural destabilization by low amounts of chlorophyll a

The present study shows that small admixtures of one chlorophyll a (Chla) molecule per several hundred lipid molecules have strong destabilizing effect on lipid bilayers. This effect is clearly displayed in the properties of the L_α-H_II transformations and results from a Chla preference for the H_II relative to the L_α phase. Chla disfavors the lamellar liquid crystalline phase L_α and induces its replacement with inverted hexagonal phase H_II, as is consistently demonstrated by DSC and X-ray diffraction measurements on phosphatidylethanolamine (PE) dispersions. Chla lowers the L_α-H_II transition temperature (42 °C) of the fully hydrated dipalmitoleoyl PE (DPoPE) by ∼ 8 °C and ∼ 17 °C at Chla/DPoPE molar ratios of 1:500 and 1:100, respectively. Similar Chla effect was recorded also for dielaidoyl PE dispersions. The lowering of the transition temperature and the accompanying significant loss of transition cooperativity reflect the Chla repartitioning and preference for the H_II phase. The reduction of the H_II phase lattice constant in the presence of Chla is an indication that Chla favors H_II phase formation by decreasing the radius of spontaneous monolayer curvature, and not by filling up the interstitial spaces between the H_II phase cylinders. The observed Chla preference for H_II phase and the substantial bilayer destabilization in the vicinity of a bilayer-to-nonbilayer phase transformation caused by low Chla concentrations can be of interest as a potential regulatory or membrane-damaging factor.     

Suppression of allergen-induced respiratory dysfunction and airway inflammation in sensitized guinea pigs by MnII(Me2DO2A), a novel superoxide scavenger compound

Reactive oxygen species produced during allergic inflammation are key players of the pathophysiology of asthma, leading to oxidative tissue injury and inactivation of endogenous manganese superoxide dismutase (MnSOD). On this ground, removal of excess superoxide anion by scavenger molecules would be beneficial and protective. Here we show that a novel manganese(II)-containing polyamine-polycarboxylic compound, termed Mn^II(Me₂DO2A), with potent superoxide dismuting properties decreases the respiratory and histopathological lung abnormalities due to allergen inhalation in a model of ovalbumin (OA)-induced allergic asthma-like reaction in sensitized guinea pigs.Severe respiratory dysfunction in response to OA aerosolic challenge arose rapidly in the sensitized animals and was accompanied by bronchoconstriction, alveolar hyperinflation, mast cell activation, increased leukocyte infiltration (evaluated by myeloperoxidase assay), oxidative lung tissue injury (evaluated by the thiobarbituric-acid-reactive substances and nitrotyrosine immunostaining), decay of endogenous MnSOD activity, production of pro-inflammatory prostaglandins, and lung cell apoptosis. Treatment with Mn^II(Me₂DO2A) (15 mg/kg, given 1 h before allergen challenge), but not the inactive congener Zn^II(Me₂DO2A) lacking redox-active metal site, significantly attenuated all the above functional, histopathological and biochemical parameters of allergic inflammation and restored the levels of MnSOD activity. In conclusion, our findings support the potential therapeutic use of Mn^II(Me₂DO2A) as novel superoxide scavenger drug in asthma and anaphylactic reactions.     

The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn^II, Cu^I, and Cd^II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β_E-domain of the early cysteine-labeled protein (E_c-1) from wheat (β_E-E_c-1), which represents the first three-dimensional structure of a plant MT. The β_E-domain comprises the 51 C-terminal residues of E_c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn^II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn^II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by ¹⁵N relaxation experiments support the results of the structure determination in that β_E-E_c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn^II with Cd^II ions and examined the effects of metal ion release on incubation with a metal ion chelator.     

The Conformer Substates of Nonoriented B-type DNA in Double Helical Poly(dG-dC)

Abstract

A nonoriented hydrated film of poly(dG-dC) with ≈20 water molecules per nucleotide (called B by Loprete and Hartman (Biochem. 32, 4077–4082 (1993)) was studied by Fourier transform infrared (FT-IR) spectroscopy either as equilibrated sample between 290 and 270 K or, after quenching into the glassy state, as nonequilibrated film isothermally at 200 and 220 K. IR spectral changes on isothermal relaxation at 200 and 220 K, caused by interconversion of two conformer substates, are revealed by difference spectra. Comparison with difference curves obtained in the same manner from two classical B-DNA forms, namely the d(CGCGAATTCGCG)₂ dodecamer and polymeric NaDNA from salmon testes, revealed that the spectral changes on B_Ito-B_II interconversion in the classical B-DNA forms are very similar to those in the B-form, and that the spectroscopic differences between the B_I and B_II features from classical B-DNA and those from the modified B-form are minor. Nonexponential kinetics of the B_I→B_II transition in the B-form of poly(dG-dC) at 200 K showed that the structural relaxation time is about three times of that in the classical B-DNA forms (≈30 versus ≈10 min at 200 K). The unexpected reversal of conformer substates interconversion (that is B_II→B_I transition on cooling from 290 K and B_I→B_II transition on isothermal relaxation at 200 K) observed for classical B-DNA occurs also in the modified B-form. We therefore conclude that restructuring of hydration shells rules the low-temperature dynamics of the B-form via its two conformer substates in the same manner reported for classical B-DNA by Pichler et al. (J. Phys. Chem. B 106, 3263–3274 (2002)).