Altered specificity in DNA binding by the lac repressor: a mutant lac headpiece that mimics the gal repressor

Recognition of the lac operator by the lac repressor involves specific interactions between residues in the repressor's recognition helix and bases in the DNA major groove. Tyr17 and Gln18, at positions 1 and 2 in the lac repressor recognition helix, can be exchanged for other amino acids to generate mutant repressors that display altered specificity. We have solved the solution structure of a protein-DNA complex of an altered-specificity mutant lac headpiece in which Tyr17 and Gln18 were exchanged for valine and alanine, respectively, as found in the recognition helix of the gal repressor. As previously described by Lehming et al. (EMBO J. 1987, 6, 3145-3153), this altered-specificity mutant of the lac repressor recognizes a variant lac operator that is similar to the gal operator Oe. The mutant lac headpiece showed the predicted specificity and is also able to mimic the gal repressor by recognizing and bending the natural gal operator Oe. These structural data show that, while most of the anchoring points that help the lac headpiece to assemble on the lac operator were preserved, a different network of protein-DNA interactions connecting Ala17 and Val18 to bases in the DNA major groove drives the specificity towards the altered operator.     

Polyaza crown ethers as non-nucleosidic building blocks in DNA conjugates: synthesis and remarkable stabilization of dsDNA

The synthesis of amphiphilic polyaza crown ether monomers X (benzyl-substituted), Y (palmityl-substituted) and Z (cholesteryl-substituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis in different alkaline metal buffer solutions. Thermal-denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y and Z) with different overall lipophilicities showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer. Multiple incorporations of benzyl-substituted polyaza crown ether monomer X as dangling ends on both sides of dsDNA resulted in strongly increased stability of the corresponding duplex.     

Modular chemistry: secondary building units as a basis for the design of highly porous and robust metal-organic carboxylate frameworks

Secondary building units (SBUs) are molecular complexes and cluster entities in which ligand coordination modes and metal coordination environments can be utilized in the transformation of these fragments into extended porous networks using polytopic linkers (1,4-benzenedicarboxylate, 1,3,5,7-adamantanetetracarboxylate, etc.). Consideration of the geometric and chemical attributes of the SBUs and linkers leads to prediction of the framework topology, and in turn to the design and synthesis of a new class of porous materials with robust structures and high porosity.     

Acetylenes with multiple triple bonds: A group of versatile An-type building blocks for the construction of functional hyperbranched polymers

AB_n-type monomers have been widely used for the synthesis of hyperbranched polymers. These monomers, however, suffer from the problems associated with the tendency of their mutually reactive A and B functional groups toward self-oligomerization. We have explored the possibility of synthesizing hyperbranched polymers using A_n-type monomers, which are stable and easy to prepare and handle, with some being even commercially available. In particular, we have tried to open new synthetic routes to hyperbranched polymers using diynes and triynes as monomers. We have developed metallic [TaBr₅, Cp^∗Ru(PPh₃)₂Cl, etc.] and nonmetallic catalysts (piperidine, DMF, etc.) for polycyclotrimerization, polycycloaddition and polycoupling of the acetylenic monomers. We have synthesized a variety of new hyperbranched polymers including polyarylenes, polytriazoles and polydiynes with high molecular weights and excellent solubility in high yields. The polymers exhibit an array of functional properties such as sensitive photonic response, high light refractivity, large optical nonlinearity, high thermal stability, strong optical limiting power and unusual aggregation-enhanced light emission. Utilizing these unique properties, we succeeded in generating fluorescent images, honeycomb patterns, polymer nanotubes, ferromagnetic ceramics, and nanoparticle catalysts.     

The mild thiolation: a useful tool for the synthesis of activated building blocks

The “mild thiolation” is a term coined specifically for the oxidation of suitable CH-acidic substrates with elemental sulfur in the presence of a base and a dipolar aprotic solvent. Dithiocarboxylates are formed under mild reaction conditions as products which, due to their increased reactivity toward nucleophiles, allow various transformations. The focus of this work is particularly the class of thioamides and their great benefits for the synthesis of heterocycles.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains werestudied through the determination of their melting points inthermodenaturation experiments. The melting point of a VH domainoriginating from a synthetic library of human VHs, which hadbeen optimized for the use as small recognition units throughthe mimicking of camelid antibody heavy chains occurring naturallywithout light chain, was 56.6C compared with 71.2C of theoriginal human VH. Its stability was improved (melting point61.6C) through three mutations to mimic camelid VHs even further:Va137 was replaced by phenylalanine and two cysteines were introducedat positions 33 and 100b. The resulting VH folded properly andformed a second intradomain disulphide between the extra cysteines.The new mutations were then built constitutively into a phage-displayVH library, from which antigen-specific VHs were selected. Twowere analysed for stability with melting points of 72.6 and75.3C. Thus secondary camelization enabled the isolation ofVHs with improved folding stabilities exceeding even that ofthe original human VH. This indicates an effect on folding stabilityfor some mutations specific in the light chain lacking camelidheavy chains.     

Molecular design of transition metal alkynyl complexes as building blocks for luminescent metal-based materials: structural and photophysical aspects

In this Account, the design and successful isolation of a series of soluble di- and polynuclear alkynyl complexes of selected metals with d(8) and d(10) electronic configurations are described. These organometallic complexes are found to exhibit rich photophysical and photochemical properties that are unique to the presence of the alkynyl ligand. By a systematic variation of the nature of the ligands, the metal centers, and certain structural features, the photophysical properties could be readily varied and their spectroscopic origins elucidated. Some of these complexes have been shown to be ideal building blocks for the design of luminescent organometallic oligomers and metal-based functional materials.     

Molecular recognition of T:G mismatched base pairs in DNA as studied by electrospray ionization mass spectrometry

Postreplicative mismatch repair (MMR) is a cellular system involved in the recognition and correction of DNA polymerase errors that escape detection in proofreading. Of the various mismatched bases, T:G pairing in DNA is one of the more common mutations leading to the formation of tumors in humans. In addition, the absence of the MMR system can generate resistance to several chemotherapeutic agents, particularly DNA-damaging substances. The main purpose of this study was the setup and validation of an electrospray ionization (ESI) mass spectrometry method for the identification of small molecules that are able to recognize T:G mismatches in DNA targets. These findings could be useful for the discovery of new antitumor drugs. The analytical method is based on the ability of electrospray to preserve the noncovalent adducts present in solution and transfer them to the gas phase. Lexitropsin derivatives (polyimidazole compounds) have been previously described as selective for T:G mismatch binding by NMR and ITC studies. We synthesized and tested various polyimidazole derivatives, one of which in particular (NMS-057) showed a higher affinity for an oligonucleotide DNA sequence containing a T:G mismatched base pair. To rationalize these findings, molecular docking studies were performed using available NMR structures. Moreover, ESI-MS experiments, performed on an orbitrap mass spectrometer, highlighted the formation of heterodimeric complexes between DNA sequences, distamycin A, and polyimidazole compounds. Our results confirm that this ESI method could be a valuable tool for the identification of new molecules able to specifically recognize T:G mismatched base pairs.     

A catalytic route to acyclic chiral building blocks: Applications of the catalytic asymmetric conjugate addition of organozinc reagents to cyclic enones

Through the Cu-phosphoramidite-catalyzed asymmetric conjugate addition a number of chiral cyclic enones are available with high ee. Here we report the sequential conjugate addition to these enones as a route towards multisubstituted chiral cyclic ketones. A subsequent Baeyer—Villiger oxidation followed by ring-opening results in various linear synthons containing multiple stereocenters. This procedure represents a short, catalytic, and highly enantioselective route to a variety of acyclic chiral building blocks.     

The "texas-sized" molecular box: a versatile building block for the construction of anion-directed mechanically interlocked structures

Over the last two decades, researchers have focused on the synthesis and development of mechanically interlocked molecules (MIMs). The intramolecular motion of mechanical bonds and the ability to induce this effect with the choice of the proper external stimuli has prompted the development of macromolecular systems that possess the ability to "perform work" at the molecular level. Currently, researchers are working to incorporate interlocked species into complex structural systems, such as molecular frameworks and nanoparticles, and to create ever more elegant noncovalent architectures. This effort provides an incentive to generate new building blocks for the construction of MIMs. In this Account, we describe progress in the development of a new cationic building block inspired by the "blue box" of Stoddart and collaborators. The blue box (cylcobis(paraquat-p-phenylene) or CBPQT(4+)) is a tetracationic, electron-deficient macrocycle widely recognized for its role in the construction of MIMs. This venerable receptor displays a high affinity for a variety of π-donor guests, and researchers have used them to construct a wide range of molecular and supramolecular structures, including rotaxanes, catenanes, pseudorotaxanes, polypseudorotaxanes, pseudo[n]polyrotaxanes, and electrochemically switchable molecules. To date, several synthetic analogues of the basic CBPQT(4+) structure have been reported, including systems containing biphenylene linkers and chiral tetracationic cyclophanes. However, researchers have not yet fully generalized the promise of the blue box. In this Account, we chronicle the development of a larger, more flexible tetracationic macrocycle, referred to as the "Texas-sized" molecular box. To highlight its relatively increased size and to distinguish it from CBPQT(4+), we have chosen to color this new receptor burnt orange. The Texas-sized box (cyclo[2](2,6-di(1H-imidazol-1-yl)pyridine)[2](1,4-dimethylenebenzene), 1(4+)·4PF(6)(-)) acts as a dynamic molecular receptor that displays an ability to adjust its shape and conformation to accommodate anionic guests of different size and charge within its central core. The use of different guests can favor different binding modes and promote the formation of different macromolecular aggregates. Furthermore, the proper selection of the guest allows for the "turning on" or "turning off" of molecular threading and can be used to produce new kinds of threaded species. This dynamic behavior is a special feature of the Texas-sized molecular box, as is its ability to stabilize a range of polypseudorotaxanes, rotaxane-containing metal-organic frameworks (MORFs), and rotaxane-based supramolecular organic frameworks (RSOFs).     

Chemical assessment of the electric arc furnace slag as construction material: Expansive compounds

To carry out a correct management of electric arc furnace slags (EAS) requires a detailed knowledge of their properties. From a chemical point of view, the EAS are surrounded by some chemical problems related to its volume instability when they are used as construction materials, due to the presence of potentially expansive compounds such as free lime and mainly free MgO. The current work focuses on the chemical assessment of the black slags coming from an electric arc furnace in order to know the possible chemical disadvantages as construction materials. A complete study of chemical and mineralogical compositions, pozzolanic activity and the quantification of main expansive compounds in EAS were realised. The results show that the nature of these slags has a very high crystallinity; total absence of pozzolanic activity and the presence of expansive compounds in slags (Cl⁻, SO₃, free CaO and free MgO) were very low, if not null concentrations.     

Zirconium nano building blocks based on the 3‐butynoic acid ligand: Synthesis and thermomechanical studies of the resulting inorganic–organic hybrid material

A new zirconium nano building block (ZrNBB) was prepared by the reaction of Zirconium tetrapropoxide with 3‐butynoic acid in an alkoxide/acid at a 1 : 3 or 1 : 6 molar ratio. The complex was characterized by FTIR, ¹H‐NMR and ¹³C‐NMR spectroscopy, thermogravimetric analysis, differential scanning calorimetry, energy‐dispersive X‐ray analysis, and elementary analysis. A spontaneous alkyne‐to‐allene isomerization process was observed for ZrNBB. A bulk sample was also prepared and analyzed via dynamical mechanical spectroscopy in compression mode. The zirconium complex was then embedded in a polymeric matrix by radical copolymerization with vinyl trimethoxysilane. The inorganic–organic hybrid material was chemically and thermally stable. The shear storage modulus and loss modulus were measured to determine the mechanical properties of the material. The preliminary results are presented. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

The eighth sentences shpuld read as follows. rFosLZ–GSTweakly associates beyond a dimer (K_a –4x10⁴ M^–1)and rJunLZ–GST associates indefinitely (K^a –4x10⁵M^–1), consistent with an isodesmic model of association.     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDS–PAGE ‘snapshot’ analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZ–GST::rFosLZ–GSTheterodimers whereas rJunLZ–GST::rFosLZ and rJunLZ::rFosLZ–GSTformed readily. Furthermore, rJunLZ–GST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (K_a >10⁶ M^-1) with no higher multimeric forms. rFosLZ–GST weaklyassociates beyond a dimer (K_a {small tilde}4x10⁵ M^-1) and rJunLZ–GSTassociates indefinitely (K_a {small tilde}4x10⁶ M^-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired.     

Molecular tweezers as synthetic receptors: molecular recognition of cationic substrates; an insight into the mechanism of complexation

The results of ¹H NMR binding studies of mole-cular tweezers 1a with various aliphatic and aromatic cations in organic solvents are described. By the use of the viologene substrates 11 and 12 bearing bulky endgroups and therefore having a dumb-bell topology it could be demonstrated that besides the obvious mechanism of complexation in which the guest molecule enters the tweezer's cavity through the open sides of the host, a second mechanism is available in which the substrate enters the cavity through a gap emerging after a substantial spreading of the tweezer's tips by at least of about 280 pm.     

A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119- Arg120-Pro121 by Gly-Gly-Gly

D-amino acid aminotransferase (EC 2.6.1.21) catalyzes the interconversion of various D-amino acids and 2-oxo acids. Each homodimer subunit consists of two domains, which are connected by a single loop, Asn118-Pro119-Arg120-Pro121. The loop has no direct contact with the active site region or the cofactor, pyridoxal 5'- phosphate. We attempted to increase the conformational flexibility of this loop through a triple glycine substitution. The resultant mutant P119G-R120G-P121G has features clearly different from the wild-type enzyme under overall as well as half-reaction conditions. The pre- steady-state kinetic analyses of half reactions showed that the mutant enzyme has kmax values higher than the wild-type enzyme towards most D- amino acids examined. A concomitant decrease in substrate affinity (1/Kd), particularly for acidic amino acids, was also observed. A putative binding site for the distal carboxyl group of acidic amino acids in the wild-type enzyme was incidentally displaced by the loop mutation, indicating a functional linkage between the interdomain loop and the active site region. This study has exemplified the usefulness of engineering relatively distant loops as a means to modify substrate specificity of an enzyme.