A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs.     

Metal Ion Dependence of Cooperative Collapse Transitions in RNA

Positively charged counterions drive RNA molecules into compact configurations that lead to their biologically active structures. To understand how the valence and size of the cations influences the collapse transition in RNA, small-angle X-ray scattering was used to follow the decrease in the radius of gyration (R_g) of the Azoarcus and Tetrahymena ribozymes in different cations. Small, multivalent cations induced the collapse of both ribozymes more efficiently than did monovalent ions. Thus, the cooperativity of the collapse transition depends on the counterion charge density. Singular value decomposition of the scattering curves showed that folding of the smaller and more thermostable Azoarcus ribozyme is well described by two components, whereas collapse of the larger Tetrahymena ribozyme involves at least one intermediate. The ion-dependent persistence length, extracted from the distance distribution of the scattering vectors, shows that the Azoarcus ribozyme is less flexible at the midpoint of transition in low-charge-density ions than in high-charge-density ions. We conclude that the formation of sequence-specific tertiary interactions in the Azoarcus ribozyme overlaps with neutralization of the phosphate charge, while tertiary folding of the Tetrahymena ribozyme requires additional counterions. Thus, the stability of the RNA structure determines its sensitivity to the valence and size of the counterions.     

Accommodation of Ca(II) ions for catalytic activity by a group I ribozyme

The wildtype Tetrahymena ribozyme cannot catalyze detectable levels of phosphotransfer activity in vitro on an exogenous RNA substrate oligonucleotide when calcium(II) is supplied as the only available divalent ion. Nevertheless, low-error mutants of this ribozyme have been acquired through directed evolution that do have activity in 10 mM CaCl₂. The mechanisms for such Ca(II) accommodation are not known. Here, we assayed the entire molecule in an effort to identify the roles of the mutations in allowing catalytic activity in Ca(II). We used four biochemical probing techniques - native-gel electrophoresis, hydroxyl radical footprinting, terbium(III) cleavage footprinting, and phosphorothioate interference mapping - to compare the solution structure of the wildtype ribozyme with that of a Ca(II)-active five-site mutant. We compared the gross folding patterns and specific metal-binding sites in both MgCl₂ and CaCl₂ solutions. We detected no large-scale folding differences between the two RNAs in either metal. However, we did discover a limited number of local folding differences, involving regions of the RNA affected by positions 42, 188, and 270. These data support the notion that Ca(II) is accommodated by the Tetrahymena ribozyme by a slight breathing at the active site, but that alterations at, near to, and distal from the active site can all contribute to Ca(II)-based activity.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.     

An active site rearrangement within the Tetrahymena group I ribozyme releases nonproductive interactions and allows formation of catalytic interactions

Biological catalysis hinges on the precise structural integrity of an active site that binds and transforms its substrates and meeting this requirement presents a unique challenge for RNA enzymes. Functional RNAs, including ribozymes, fold into their active conformations within rugged energy landscapes that often contain misfolded conformers. Here we uncover and characterize one such “off-pathway” species within an active site after overall folding of the ribozyme is complete. The Tetrahymena group I ribozyme (E) catalyzes cleavage of an oligonucleotide substrate (S) by an exogenous guanosine (G) cofactor. We tested whether specific catalytic interactions with G are present in the preceding E•S•G and E•G ground-state complexes. We monitored interactions with G via the effects of 2′- and 3′-deoxy (–H) and −amino (–NH₂) substitutions on G binding. These and prior results reveal that G is bound in an inactive configuration within E•G, with the nucleophilic 3′-OH making a nonproductive interaction with an active site metal ion termed M_A and with the adjacent 2′-OH making no interaction. Upon S binding, a rearrangement occurs that allows both –OH groups to contact a different active site metal ion, termed M_C, to make what are likely to be their catalytic interactions. The reactive phosphoryl group on S promotes this change, presumably by repositioning the metal ions with respect to G. This conformational transition demonstrates local rearrangements within an otherwise folded RNA, underscoring RNA''s difficulty in specifying a unique conformation and highlighting Nature''s potential to use local transitions of RNA in complex function.     

Side-on binding of p-sulphonatocalix[4]arene to the dinuclear platinum complex trans-[{PtCl(NH3)2}2μ-dpzm] and its implications for anticancer drug delivery

The utility of p-sulphonatocalix[4]arene (s-CX[4]) as a drug delivery vehicle for multinuclear platinum anticancer agents, using trans-[{PtCl(NH₃)₂}₂μ-dpzm]²⁺ (di-Pt; where dpzm = 4,4′-dipyrazolylmethane) as a model complex, has been examined using ¹H nuclear magnetic resonance, electrospray ionisation mass spectrometry, molecular modelling and in vitro growth inhibition assays. s-CX[4] binds di-Pt in a side-on fashion in a ratio of 1:1, with the dpzm ligand of the metal complex located within the s-CX[4] cavity with binding further stabilised by ion-ion interactions and hydrogen bonding between the metal complex am(m)ine groups and the s-CX[4] sulphate groups. Partial encapsulation of di-Pt within the cavity does not prevent binding of 5′-guanosine monophosphate to the metal complex. When bound to two individual guanosine molecules, di-Pt also remains partially bound by s-CX[4]. The cytotoxicity of free di-Pt and s-CX[4] and their host guest complex was examined using in vitro growth inhibition assays in the A2780 and A2780cis human ovarian cancer cell lines. Free di-Pt has an IC₅₀ of 100 and 60 μM, respectively, in the cell lines, which is significantly less active than cisplatin (1.9 and 8.1 μM, respectively). s-CX[4] displays no cytotoxicity at concentrations up to 1.5 mM and does not affect the cytotoxicity of di-Pt, probably because its low binding constant to the metal complex (6.8 × 10⁴ M⁻¹) means the host-guest complex is mostly disassociated at biologically relevant concentrations.     

Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A

The crystal structures of an aspartic proteinase from Trichoderma reesei (TrAsP) and of its complex with a competitive inhibitor, pepstatin A, were solved and refined to crystallographic R-factors of 17.9% (R_free = 21.2%) at 1.70 Å resolution and 15.8% (R_free = 19.2%) at 1.85 Å resolution, respectively. The three-dimensional structure of TrAsP is similar to structures of other members of the pepsin-like family of aspartic proteinases. Each molecule is folded in a predominantly β-sheet bilobal structure with the N-terminal and C-terminal domains of about the same size. Structural comparison of the native structure and the TrAsP-pepstatin complex reveals that the enzyme undergoes an induced-fit, rigid-body movement upon inhibitor binding, with the N-terminal and C-terminal lobes tightly enclosing the inhibitor. Upon recognition and binding of pepstatin A, amino acid residues of the enzyme active site form a number of short hydrogen bonds to the inhibitor that may play an important role in the mechanism of catalysis and inhibition. The structures of TrAsP were used as a template for performing statistical coupling analysis of the aspartic protease family. This approach permitted, for the first time, the identification of a network of structurally linked residues putatively mediating conformational changes relevant to the function of this family of enzymes. Statistical coupling analysis reveals coevolved continuous clusters of amino acid residues that extend from the active site into the hydrophobic cores of each of the two domains and include amino acid residues from the flap regions, highlighting the importance of these parts of the protein for its enzymatic activity.     

Acid dissociation of 2,2′-dipyridylamine in non-aqueous medium when chelated to some Ru(II)N4 cores

[Ru(2,2′-bipyridine)₂(Hdpa)](BF₄)₂ · 2H₂O (1), [Ru(1,10-phenanthroline)₂(Hdpa)] (PF₆)₂ · CH₂Cl₂ (2) and [Ru(4,4,4′,4′-tetramethyl-2,2′- bisoxazoline)₂(Hdpa)] (PF₆)₂ (3) are synthesized where Hdpa is 2,2′-dipyridylamine. The X-ray crystal structures of 1 and 2 have been determined. Hdpa in 1 and 2 is found to bind the metal via the two pyridyl N ends. Comparing the NMR spectra in DMSO-d₆, it is concluded that 3 has a similar structure. The pK_a values (for the dissociation of the NH proton in Hdpa) of free Hdpa and its complexes are determined in acetonitrile by exploiting molar conductance. These correlate linearly with the chemical shift of the NH proton in the respective entities.     

Crystal structure and fluorescence of a europium (III) complex: EuCl3(2,2′-bipyridine N,N dioxide) · 2CH3OH

We report the synthesis and characterization of a seven coordinate europium complex, [EuCl₃(C₁₀H₈N₂O₂) ·  2CH₃OH]. The growing interest in developing efficient light conversion molecular devices (LCMDs) necessitates the need for new fluorescent materials. Ideal physicochemical properties of the materials include ligand absorption, efficient metal to ligand transfer, and strong luminescence with a relatively long decay time. The design of such material requires distinct absorbing (ligand) and emitting (metal ion) components. While Eu³⁺ cation has a non-degenerate emitting level, 2,2′-bipyridine N,N dioxide is a heterocyclic ligand known to exhibit strong luminescence. Additional characterization is also described, including single crystal X-ray diffraction, IR and UV-Vis spectroscopies and elemental analysis.     

Synthesis, crystal structure and luminescent behaviour of coordination complexes of copper with bi- and tridentate amines and phosphonic acids

The synthesis and crystal structure of four new copper(I) and copper(II) supramolecular amine, and amine phosphonate, complexes is reported. Reaction of copper(I) with 2-,9-dimethyl-1-10-phenanthroline (dmp) produced a stable 4-coordinate Cu(I) species, [Cu^(I)(dmp)₂]Cl · MeOH · 5H₂O (2), i.e., the increased steric hindrance in the ‘bite’ area of dmp did not prevent interaction with the metal and provided protection against oxidation which was not possible for the phen analogue [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36]. Subsequent addition of phenylphosphonic acid to (2) produced two structures from alternative synthetic routes. An ‘in situ’ process yielded red block Cu(I) crystals, [Cu^(I)(dmp)₂] · [C₆H₅PO₃H₂ · C₆H₅PO₃H] (4), whilst recrystallisation of (2) prior to addition of the acid (‘stepwise’ process) produced a green, needle-like Cu(II) complex, [Cu^(II)(dmp) · (H₂O)₂ · C₆H₅PO₂(OH)] [C₆H₅PO₂(OH)] (3). However, addition of excess dmp during the ‘stepwise’ process forced the equilibrium towards product (4) and resulted in an optimum yield (99%). The structure of (4) was similar to the phen analogue, [Cu^(II)Cl(phen)₂] · [C₆H₅PO₂(OH) · C₆H₅PO(OH)₂] (1) [R. Clarke, K. Latham, C. Rix, M. Hobday, J. White, CrystEngCommun. 7(3) (2005), 28-36], but the presence of dmp exerted some influence on global packing, whilst (3) exists as a polymeric layered material. In contrast, reaction of copper(I) with di-2-pyridyl ketone (dpk), followed by phenylphosphonic acid produced purple/blue Cu(II) species, [Cu^(II)(dpk · H₂O)₂] Cl₂ · 4H₂O (5), and [Cu^(II)(dpk · H₂O)₂] · [C₆H₅PO₂(OH)₂ · C₆H₅PO(OH)₂] (6), respectively, i.e., in both cases oxidation of copper occurred. Solid-state luminescence was observed in (2) and (4). The latter showing a 5-fold enhancement in intensity.     

Preparation and physical properties of chitin fatty acids esters

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 °C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T_g) and the tensile modulus (E′ at 25 °C) are highest for chitin acetate (218 °C and 5.8 GPa), and lowest for chitin octanoate (182 °C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T_g and the E′ decrease with an increase in the chain length of the acyl constituent.     

The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

Synthesis, structure and redox properties of an unexpected trinuclear copper(II) complex with aspartame: [Cu(apm)2Cu(μ-N,O:O′-apm)2(H2O)Cu(apm)2(H2O)] · 5H2O

Structural, magnetic and spectroscopic data of a new trinuclear copper(II) complex with the ligand aspartame (apm) are described. [Cu(apm)₂Cu(μ-N,O:O′-apm)₂(H₂O)Cu(apm)₂(H₂O)] · 5H₂O crystallizes in the triclinic system, space group P1 (#1) with a = 7.3300(1) Å, b = 15.6840(1) Å, c = 21.5280(1) Å, α = 93.02(1)°, β = 93.21(1)°, γ = 92.66(1)° and Z = 1. Aspartame coordinates to Cu(II) through the carboxylate and β-amino groups. The carboxylate groups of the two central ligands act as bidentate bridges in a syn-anti conformation while the carboxylate groups of the four peripheral ligands are monodentate in a syn conformation. The central copper ion is in a distorted square pyramidal geometry with the apical position being occupied by one oxygen atom of the water molecule. The two terminal copper(II) atoms are coordinated to the ligands in the same position but their coordination sphere differs from each other due to the fact that one copper atom has a water molecule in an apical position leading to an octahedral coordination sphere while the other copper atom is exclusively coordinated to aspartame ligands forming a distorted square pyramidal coordination sphere. Thermal analysis is consistent with the X-ray structure. EPR spectra and CV curves indicate a rupture of the trinuclear framework when this complex is dissolved in ethanol or DMF, forming a mononuclear species, with a tetragonal structure.     

Synthesis and crystal structure of one-dimensional zinc(II) polymer constructed by salicylate and trans-1,2-bis(4-pyridyl)ethylene

A new zinc(II) compound, [Zn₂(Hsal)₄(4,4′-bpe)₂] · [Zn(Hsal)₂(4,4′-bpe)(DMF)(H₂O)] · CH₃OH (1) (Hsal = salicylate and 4,4′-bpe = trans-1,2-bis(4-pyridyl)ethylene) has been synthesized and characterized by elemental analysis, infrared spectroscopy, thermogravimetric analysis, and single crystal X-ray diffraction. The crystal structure consists of three independent moieties: [Zn₂(Hsal)₄(4,4′-bpe)₂], [Zn(Hsal)₂(4,4′-bpe)(DMF)(H₂O)], and non-coordinated CH₃OH molecule. In the compound two independent moieties which are connected by 4,4′-bpe to form 1-D chains, respectively, are further expended to accomplish 2-D network through hydrogen-bonding interactions between non-coordinated methanol and coordinated water molecule or carboxylate oxygen atoms of Hsal ligands.     

Discrete and 1D coordination polymeric chloro-bridged copper(II) dimers exhibiting ferro- and antiferromagnetic exchange coupling: Magneto-structural correlations and non-covalent interactions

The synthesis and characterization of two 1D coordination polymers [Cu₂(MHL)Cl₂][ClO₄]₂ · CH₃CN · THF (2 · CH₃CN · THF) and [Cu₂(MPyPz)Cl₂][ClO₄]₂ · CH₃CN (3 · CH₃CN), having repetitive units, of m-xylyl-based ligands with terminal tridentate (2-pyridyl)alkylamine (MHL = α,α′-bis[N-(2-pyridylethyl)-N-(2-pyridylmethyl)amino]-m-xylene) and (2-pyridyl)alkylamine/pyrazole (MPyPz = α,α′-bis[N-(2-pyridylethyl)-N-(pyrazol-1-ylmethyl)amino]-m-xylene) coordination have been accomplished. X-ray crystallographic studies reveal that the copper(II) centers in the recently reported dichloro-bridged discrete complex [{Cu(MeL)Cl}₂][ClO₄]₂ (1) of a tridentate (2-pyridyl)alkylamine ligand [MeL = methyl[2-(2-pyridyl)ethyl](2-pyridylmethyl)amine], 2 · CH₃CN · THF, and 3 · CH₃CN have distorted square-pyramidal geometry, sharing a base-to-apex edge with parallel basal planes. Variable-temperature susceptibility measurements in the range of 2-300 K reveal antiferromagnetic for 1 [J (singlet-triplet energy gap) = −3.89 cm⁻¹] and 2 · CH₃CN · THF (J = −1.84 cm⁻¹), and ferromagnetic for 3 · CH₃CN (J = +6.27 cm⁻¹) coupling. The complexes provide useful information for the magneto-structural correlations.     

The structural definition of adducts of stoichiometry MX:dpex (2:3)(∞), M = Cu, Ag, X = simple anion, dpex = Ph2E(CH2)xEPh2, E = P, As

Single crystal X-ray structural characterizations are recorded for a number of adducts of MX:dpex (2:3) stoichiometry (MX = simple univalent copper or silver salt; dpex = Ph₂E(CH₂)_xEPh₂ (E = P, As)). CuX:dppe (2:3) (X = Cl, Br, I, CN) are binuclear [(dppe-P,P′)CuX(P-dppe-P′)CuX(P,P′-dppe)], all centrosymmetric. AgX:dpex (2:3) (dpex = ‘dpae’ (Ph₂As(CH₂)₂AsPh₂), X = Br, F₃CCO₂ (= ‘tfa’), F₃CSO₃ (≡ ‘tfs’); dpex = ‘dpape’ (Ph₂As(CH₂)₂PPh₂), X = CN, SCN, OClO₃) are one-dimensional polymers ?-E′)₁AgX(E-dpex-E′)₂-AgX(E-dpex-E′)₁AgX?, P, As sites scrambled in the latter. AgNO₃:dpam (2:3) is also a one-dimensional polymer, ?AgO·NO·OAg(As-dpam-As)AgO·NO·OAg? (‘dpam’ ≡ Ph₂As(CH₂)₂AsPh₂). AgX:dpae (2:3) (X = I, CN, ClO₄, NO₃) and AgX:dpape (2:3) (X = Br, I, NO₃) are two-dimensional polymers with large 30-membered macrocyclic rings; similar webs are found for dppx ligands in AgOH:dppb (2:3) and AgNCO, Agtfa:dpph (2:3) with 42- and 54-membered rings. Complexes AgX:dpape (1:3) (X = Cl, Br) are defined as mono-nuclear [XAg(Ph₂P(CH₂)₂AsPh₂)₃] arrays, the unidentate ligands predominantly P-bound. Synthetic procedures for the adducts are reported, selected compounds being characterized both in solution (¹H, ³¹P NMR, ESI MS) and in the solid state (IR).     

Linkage of cation binding and folding in human telomeric quadruplex DNA

Formation of DNA quadruplexes requires monovalent cation binding. To characterize the cation binding stoichiometry and linkage between binding and folding, we carried out KCl titrations of Tel22 (d[A(GGGTTA)₃]), a model of the human telomere sequence, using a fluorescent indicator to determine [K⁺]_free and circular dichroism to assess the extent of folding. At [K⁺]_free = 5 mM (sufficient for > 95% folding), the apparent binding stoichiometry is 3K⁺/Tel22; at [K⁺]_free = 20 mM, it increased to 8-10K⁺/Tel22. Thermodynamic analysis shows that at [K⁺]_free = 5 mM, K⁺ binding contributes approximately − 4.9 kcal/mol for folding Tel22. The overall folding free energy is − 2.4 kcal/mol, indicating that there are energetically unfavorable contributions to folding. Thus, quadruplex folding is driven almost entirely by the energy of cation binding with little or no contribution from other weak molecular interactions.     

Syntheses and 1D structures of organic-inorganic hydrid polymers combining M(ClO4)2 (M = Cd, Zn) junctions and the semi-flexible bis-pyridyl ligand 3-pmpmd (N,N′-bis(3-pyridylmethyl)pyromellitic diimide)

Two 1D organic-inorganic coordination polymers, [Cd(3-pmpmd)(CH₃CN)₂(H₂O)₂]_n · 2n(ClO₄)₂ (1) and [Zn(3-pmpmd)_1.5(H₂O)₂]_n · 2n(ClO₄)₂ · nCH₃CN (2), were obtained from M(ClO₄)₂ (M = Cd, Zn) and the semi-flexible 3,3′-N-donor bis-pyridyl ligand 3-pmpmd: 1 has an 1D zigzag framework with 3-pmpmd in the Z_T-mode (anti, trans-) conformation, while 2 has an 1D rod and loop network with 3-pmpmd in both Z_T- and Z_C-mode (anti, cis-) conformations. Results showed that the metal ions could influence the coordination mode of a semi-flexible bis-pyridyl ligand.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.