Stability of folding structure of Zic zinc finger proteins

Zic family proteins have five C₂H₂-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.     

A structural approach reveals how neighbouring C2H2 zinc fingers influence DNA binding specificity

Development of an accurate protein–DNA recognition code that can predict DNA specificity from protein sequence is a central problem in biology. C₂H₂ zinc fingers constitute by far the largest family of DNA binding domains and their binding specificity has been studied intensively. However, despite decades of research, accurate prediction of DNA specificity remains elusive. A major obstacle is thought to be the inability of current methods to account for the influence of neighbouring domains. Here we show that this problem can be addressed using a structural approach: we build structural models for all C₂H₂-ZF–DNA complexes with known binding motifs and find six distinct binding modes. Each mode changes the orientation of specificity residues with respect to the DNA, thereby modulating base preference. Most importantly, the structural analysis shows that residues at the domain interface strongly and predictably influence the binding mode, and hence specificity. Accounting for predicted binding mode significantly improves prediction accuracy of predicted motifs. This new insight into the fundamental behaviour of C₂H₂-ZFs has implications for both improving the prediction of natural zinc finger-binding sites, and for prioritizing further experiments to complete the code. It also provides a new design feature for zinc finger engineering.     

DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference

[Fe₂L₃]⁴⁺ (L = C₂₅H₂₀N₄) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe₂L₃]⁴⁺, we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine–pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe₂(L-CF₃)₃]⁴⁺ and [Fe₂(L-Ph)₃]⁴⁺ results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.     

The structure of Λ-β1-[Co(R,R-picchxn)(S-tyr)]Br2 · 3.5H2O and solution configuration of Λ-β1-[Co(R,R-picchxn)(aromatic S-aminoacidate)] complexes: model systems for the investigation of non-covalent aromatic interactions

Intramolecular non-covalent interactions involving aromatic residues in the ternary species Λ-β₁-[Co(R,R-picchxn)(S-aa)]²⁺ (aa=Tyr, OMe-tyr or Phe) have been investigated. Such interactions are important to discriminatory processes associated with molecular recognition in chemical and biochemical systems. The single-crystal X-ray study of Λ-β₁-[Co(R,R-picchxn)(S-tyr)]Br₂ · 3.5H₂O demonstrates the influence of intramolecular π-π and bifurcated NH-π interactions in determining the molecular conformation of the complex cation in the solid state. The Co(III) complexes synthesised are diamagnetic, and have been fully assigned in solution using multidimensional NMR techniques. Remarkably, the solid state conformation observed for Λ-β₁-[Co(R,R-picchxn)(S-tyr)]²⁺ has been shown to predominate in solutions of all the complexes, as evidenced by appropriate rOe correlations. ¹H NMR measurements carried out in order to determine equilibrium rotamer distributions confirm the dominance of this conformer in solution. NMR measurements also show that rotamer populations are relatively unchanged at elevated temperatures and in a variety of solvents. The results of this detailed study, which demonstrate the significance of non-covalent interactions involving aromatic residues to the determination of the molecular conformation, serve to highlight the suitability of these simple ternary Co(III) complexes to act as models for such interactions.     

Structure and DNA binding characteristics of the three‐Cys2His2 domain of mouse testis zinc finger protein

The C‐terminal three‐Cys₂His₂ zinc‐finger domain (TZD) of mouse testis zinc‐finger protein binds to the 5′‐TGTACAGTGT‐3′ at the Aie1 (aurora‐C) promoter with high specificity. Interestingly, the primary sequence of TZD is unique, possessing two distinct linkers, TGEKP and GAAP, and distinct residues at presumed DNA binding sites at each finger, especially finger 3. A K_d value of ～10^?8 M was obtained from surface plasmon resonance analysis for the TZD‐DNA complex. NMR structure of the free TZD showed that each zinc finger forms a typical ββα fold. On binding to DNA, chemical shift perturbations and the R₂ transverse relaxation rate in finger 3 are significantly smaller than those in fingers 1 and 2, which indicates that the DNA binding affinity in finger 3 is weaker. Furthermore, the shift perturbations between TZD in complex with the cognate DNA and its serial mutants revealed that both ADE7 and CYT8, underlined in 5′‐ATATGTACAGTGTTAT‐3′, are critical in specific binding, and the DNA binding in finger 3 is sequence independent. Remarkably, the shift perturbations in finger 3 on the linker mutation of TZD (GAAP mutated to TGEKP) were barely detected, which further indicates that finger 3 does not play a critical role in DNA sequence‐specific recognition. The complex model showed that residues important for DNA binding are mainly located on positions ?1, 2, 3, and 6 of α‐helices in fingers 1 and 2. The DNA sequence and nonsequence‐specific bindings occurring simultaneously in TZD provide valuable information for better understanding of protein–DNA recognition. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases

The SinI and EcoRII DNA methyltransferases recognize sequences (GG^A/_TCC and CC^A/_TGG, respectively), which are characterized by an ^A/_T ambiguity. Recognition of the A·T and T·A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine·C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G·C or a C·G base pair in the center of the recognition sequence (GG^G/_CCC and CC^G/_CGG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG^A/_TCC and GG^G/_CCC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that ^A/_T versus ^G/_C discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.     

Natural isoflavones regulate the quadruplex–duplex competition in human telomeric DNA

Effects of natural isoflavones on the structural competition of human telomeric G-quadruplex d[AG₃(T₂AG₃)₃] and its related Watson–Crick duplex d[AG₃(T₂AG₃)₃-(C₃TA₂)₃C₃T] are investigated by using circular dichroism (CD), ESI-MS, fluorescence quenching measurement, CD stopped-flow kinetic experiment, UV spectroscopy and molecular modeling methods. It is intriguing to find out that isoflavones can stabilize the G-quadruplex structure but destabilize its corresponding Watson–Crick duplex and this discriminated interaction is intensified by molecular crowding environments. Kinetic experiments indicate that the dissociation rate of quadruplex (k_{obs290 nm}) is decreased by 40.3% at the daidzin/DNA molar ratio of 1.0 in K⁺, whereas in Na⁺ the observed rate constant is reduced by about 12.0%. Furthermore, glycosidic daidzin significantly induces a structural transition of the polymorphic G-quadruplex into the antiparallel conformation in K⁺. This is the first report on the recognition of isoflavones with conformational polymorphism of G-quadruplex, which suggests that natural isoflavone constituents potentially exhibit distinct regulation on the structural competition of quadruplex versus duplex in human telomeric DNA.     

Synthesis and characterization of silver(I) derivatives containing acylpyrazolonate and phosphino ligands: X-ray crystal structures of monomeric [Ag(Q)(PPh3)2] and of dimeric [{Ag(Q)(PiBu3)}2] (Q = 1-phenyl-3-methyl-4-tert-butylacetylpyrazolon-5-ato)

New silver(I) acylpyrazolonate derivatives [Ag(Q)], [Ag(Q)(PR₃)]₂ and [Ag(Q)(PR₃)₂] (HQ = 1-R¹-3-methyl-4-R²(CO)pyrazol-5-one, HQ^Bn = R¹ = C₆H₅, R² = CH₂C₆H₅; HQ^CHPh2 = R¹ = C₆H₅, R² = CH(C₆H₅)₂; HQ^nPe = R¹ = C₆H₅, R² = CH₂C(CH₃)₃; HQ^tBu = R¹ = C₆H₅, R² = C(CH₃)₃; HQ^fMe = R¹ = C₆H₄-p-CF₃, R² = CF₃; HQ^fEt = R¹ = C₆H₅, R² = CF₂CF₃; R = Ph or iBu) have been synthesized and characterized in the solid state and solution. The crystal structure of 1-(4-trifluoromethylphenyl)-3-methyl-5-pyrazolone, the precursor of proligand HQ^fMe and of derivatives [Ag(Q^nPe)(PPh₃)₂] and [Ag(Q^nPe)(PiBu₃)]₂ have been investigated. [Ag(Q^nPe)(PPh₃)₂] is a mononuclear compound with a silver atom in a tetrahedrally distorted AgO₂P₂ environment, whereas [Ag(Q^nPe)(PiBu₃)]₂ is a dinuclear compound with two O₂N-exotridentate bridging acylpyrazolonate ligands connecting both silver atoms, their coordination environment being completed by a phosphine ligand.     

Synthesis of dimethylcobalt(III) complexes containing trimethylphosphine and 2-formyl-phenolato- or 2-formyl-enolato(O:O) ligands

Substituted salicylaldehydes [C₆HR¹R²R³(CHO)(OH)] react with CoMe₃(PMe₃)₃ to afford 6-coordinate (cis-dimethyl)(2-formyl-phenolato)trans-bis(trimethylphosphine)cobalt(III) compounds Co[C₆HR¹R²R³(CHO)(O)Me₂](PMe₃)₂ (1: R¹ = H; R² = Me; R³ = tert-Bu; 2: R¹, R² = C₆H₄; R³ = H). Accordingly, substituted enolated malonic dialdehydes (CHO-CR⁴CR⁵-OH) react with CoMe₃(PMe₃)₃ to afford 6-coordinate (cis-dimethyl)(2-formyl-enolato)trans-bis(trimethylphosphine)cobalt(III) compounds Co[(CHO-CR⁴CR⁵-O)(Me)₂](PMe₃)₂ (3: R⁴, R⁵ = (CH₂)₂C₆H₄; 4: R⁴ = R⁵ = C₆H₅). In the molecular structure of 4, the cobalt atom is centred in an octahedral coordination geometry brought about by a six-membered chelate ring (O:O-ligand), cis-dimethyl and trans-trimethylphosphine groups. A reaction mechanism is suggested.     

Novel ruthenium(II) diamine complexes

The complex cations [Ru(C₇H₁₆N₂)(C₁₀H₁₄)Cl]⁺, [Ru(C₇H₁₆N₂)(C₆H₆)Cl]⁺, [Ru(C₉H₁₈N₂)(C₆H₆)Cl]⁺, [Ru(C₉H₁₈N₂)(C₁₀H₁₄)Cl]⁺ and [Ru(C₁₄H₁₆N₂)(C₁₀H₁₄)Cl]⁺ have been synthesised from the reaction between the ruthenium-arene complexes [with C₆H₆ (benzene) or C₁₀H₁₄ (p-cymene)] and the respective chiral diamines [C₇H₁₆N₂=(S)-(−)-2-aminomethyl-1-ethylpyrrolidine, C₉H₁₈N₂=(S)-(+)-2-(pyrrolidinylmethyl)-pyrrolidine, or C₁₄H₁₆N₂=(1R,2R)-(+)-1,2-diphenylethylenediamine], isolated and characterised as chloride salts using single-crystal X-ray diffraction. All complexes were fully characterised by elemental analysis, mass spectrometry, ¹³C and ¹H NMR, and also found to exhibit catalytic activity in the transfer hydrogenation of acetophenone to 1-phenylethanol at 50 °C (enantiomeric excesses range from ca. 25% to 60%, and conversions from ca. 30% to 50%).     

The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron

Hydrogen peroxide (H₂O₂) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H₂O₂ can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H₂O₂ by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that yaaA, an uncharacterized gene found in many bacteria, was induced by H₂O₂ in Escherichia coli as part of its OxyR regulon. Here we confirm that yaaA is a key element of the stress response to H₂O₂. In a catalase/peroxidase-deficient (Hpx⁻) background, yaaA deletion mutants grew poorly, filamented extensively, and lost substantial viability when they were cultured in aerobic LB medium. The results from a thyA forward mutagenesis assay and the growth defect of the yaaA deletion in a recombination-deficient (recA56) background indicated that yaaA mutants accumulated high levels of DNA damage. The growth defect of yaaA mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx⁻ yaaA cells had a higher hydroxyl radical (HO^•) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H₂O₂ stress the induction of YaaA is a critical device to suppress intracellular iron levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown.     

Cyclic and high molecular weight chiral binaphthoxy-phosphazene polymers carrying Br or trimethylsilyl-acetylene groups

The new chiral and functionalized cyclic binaphthoxyphosphazenes R,R,R-[N₃P₃(O₂C₂₀H₁₀Br₂)₃] (R-1), R,R,R-[N₃P₃(O₂C₂₀H₁₀(CCSiMe₃)₂)₃] (R-2), and the high molecular weight linear polymers R/S-[NP(O₂C₂₀H₁₀Br₂)]_n (R/S-3), R-[NP(O₂C₂₀H₁₀Br₂)]_n (R-3), and R-{NP[O₂C₂₀H₁₀(CCSiMe₃)₂]}_n, (R-4), with M_w on the order of 10⁶ and very high T_g, have been synthesized and characterized by IR and NMR spectroscopy. The optically active polymer (R-3) was configurationally stable below 300 °C, but at higher temperatures an atropisomerization process took place that became faster near the glass transition temperature (ca. 350 °C).     

Sterol-phospholipid interactions in model membranes Effect of polar group substitutions in the cholesterol side-chain at C20 and C22

The interactions of phospholipids with four different cholesterol derivatives substituted with one OH or one keto group at position C₂₀ or C₂₂ of the side-chain were studied. The derivatives were the 22,R-hydroxy; 22,S-hydroxy; 22-keto- and 20,S-hydroxycholesterol. Two aspects of the interactions were investigated: (1) the effect of the cholesterol derivatives on the gel → liquid crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) and of dielaidoylphosphatidylethanolamine (DEPE) monitored by differential scanning calorimetry and (2) The effect on the lamellar → hexagonal H_II phase transition of DEPE monitored by DSC and by ³¹P-NMR to determine structural changes. The gel → liquid crystalline phase transition was affected by the cholesterol derivatives to a much larger extent in the case of DPPC than of DEPE. In both cases, there was a differential effect of the four derivatives, the 22,R-hydroxycholesterol being the less effective. In DPPC-sterol 1:1 systems, 22,R-hydroxycholesterol does not suppress the melting transition, the ΔH values becomes 7.1 kcal · mol^?1 as compared to 8.2 kcal · mol^?1 for the pure lipid. 22,S-OH cholesterol has a much stronger effect (ΔH = 3.1 kcal · mol^?1) and 22-ketocholesterol suppresses the transition completely. In DEPE mixtures of all these compounds, the melting transition of the phospholipid is still observable. The transition temperature was shifted to lower values (?13.5°C in the presence of 20,S-OH cholesterol). The ΔH of the transition was lowered by these compounds except in DEPE-22,R-OH cholesterol mixtures and the cooperativity of the transition (reflected by the width at half peak height) was reduced. The lamellar → hexagonal H_II phase transition was also affected by the presence of these cholesterol derivatives. The transition temperature value was depressed with all these compounds. 20,S-OH cholesterol was the most effective followed by 22,R-OH cholesterol. The ΔH of the transition was not strongly affected. The molecular interfacial properties of these derivatives were studied by the monomolecular film technique. It is most likely that 22,R-OH cholesterol due to the hydroxyl groups at the 3β- and 22,R-positions orients with the sterol nucleus lying flat at the air/water interface, since the compression isotherm of either the pure sterol or the DOPC-sterol mixture (molar ration, 1:1) monomolecular film exhibits a transition at approx. 103 Ų, corresponding to the area of revolution of the sterol nucleus. This remarkable property, due probably to the existence of a kink between the side-chain and the long axis of the steroid nucleus, might explain the smaller effect of this sterol on the melting transition of either PC or PE systems.