Site-directed mutagenesis of dimethylsulfoxide reductase from Rhodobacter capsulatus: The critical roles of tyrosine-114 and tryptophan-116

The crystal structure of six functionally-distinct enzymes of the DMSO reductase family of molybdenum enzymes has revealed that the tertiary structure of the polypeptide that binds the bis(MGD)Mo cofactor is highly conserved. Differences in the catalytic properties of enzymes of this family are almost certainly dependent upon differences in the structure ofthe MO active site. In DMSO reductase from Rhodobacter species tryptophan- 116 (W 116) hydrogen-bonds to an 0x0 group coordinated to the MO ion. In addition a second amino acid side chain from tyrosine-114 (Y 114) is in close proximity to the 0x0 group. We have investigated the role of Y 114 and W 116 in DMSO reductase using site-directed mutagenesis,

Photochemical Reactions of fac-[Mn(CO)(3)(phen)imidazole](+): Evidence for Long-Lived Radical Species Intermediates

The electronic absorption spectrum of fac[Mn(CO)(3)(phen)imH](+), fac-1 in CH(2)Cl(2) is characterized by a strong absorption band at 378 nm (epsilon(max) = 3200 mol(-1) L cm(-1)). On the basis of quantum mechanical calculations, the visible absorption band has been assigned to ligand-to-ligand charge-transfer (LLCT, im -> phen) and metal-to-ligand charge-transfer (MLCT, Mn -> phen) charge transfer transition. When fac-1 in CH(2)Cl(2) is irradiated with 350 nm continuous light, the absorption features are gradually shifted to represent those of the meridional complex mer-[Mn(CO)(3)(phen)imH](+), mer-1 (lambda(max) = 556 nm). The net photoreaction under these conditions is a photoisomerization, although, the presence of the long-lived radical species was also detected by (1)H NMR and FTIR spectroscopy. 355 nm continuous photolysis of fac-1 in CH(3)CN solution also gives the long-lived intermediate which is readly trapped by metylviologen (MV(2+)) giving rise to the formation of the one-electron reduced methyl viologen (MV(center dot+)). The UV-vis spectra monitored during the slow (45 min) thermal back reaction exhibited isosbestic conversion at 426 nm. On the basis of spectroscopic techniques and quantum mechanical calculations, the role of the radicals produced is analyzed.

The 2.0 angstrom crystal structure of a pocilloporin at pH 3.5: The structural basis for the linkage between color transition and halide binding

The pocilloporin Rtms5 and an engineered variant Rtms5(H146S) undergo distinct color transitions (from blue to red to yellow to colorless) in a pH-dependent manner. pK(a) values of 4.1 and 3.2 were determined for the blue (absorption lambda(max), 590 nm) to yellow (absorption lambda(max), similar to 453 nm) transitions of Rtms5 and Rtms5H(146). The pK(a) for the blue-yellow transition of Rtms5H(146S) increased by 1.4 U in the presence of 0.1 M KI, whereas the pK(a) for the same transition of Rtms5 was relatively insensitive to added halides. To understand the structural basis for these observations, we have determined to 2.0 A resolution the crystal structure of a yellow form of Rtms5(H146S) at pH 3.5 in the presence of iodide. Iodide was found occupying a pocket in the structure with a pH of 3.5, forming van der Waals contacts with the tyrosyl moiety of the chromophore. Elsewhere, it was determined that this pocket is occupied by a water molecule in the Rtms5(H141S) structure (pH 8.0) and by the side chain of histidine 146 in the wild-type Rtms5 structure. Collectively, our data provide an explanation for the observed linkage between color transitions for Rtms5(H146S) and binding to halides.

Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.

Carbon hydroxylation of alkyltetrahydropyranols: A paradigm for spiroacetal biosynthesis in Bactrocera sp.

[GRAPHICS] In a number of Bactrocera species the penultimate step in the biosynthesis of spiroacetals is shown to be the hydroxylation of an alkyltetrahydropyranol followed by cyclization, The monooxygenases that catalyze this side chain hydroxylation show a strong preference for oxidation four carbons from the hemiketal center, to produce the spiroacetal, The hydroxy spiroacetals observed in Bactrocera appear to derive from direct oxidation of the parent spiroacetals and not from alternate precursors.

Molecular Lego (R): non-centrosymmetric alignment within interdigitating layers

(E)-N-Hexadecyl-4-[2-(4-octadecyloxynaphthyl) ethenyl] quinolinium bromide, which has a wide-bodied chromophore and terminal n-alkyl groups, adopts a U-shape when spread at the air-water interface but a stretched conformation when compressed to ca. 35 mN m(-1). The high-pressure phase has a narrow stability range prior to collapse but may be extended from 40 to 60 mN m(-1) by co-spreading the dye in a 1 : 1 ratio with docosanoic acid. The mixed Langmuir-Blodgett (LB) film has a monolayer thickness of 4.6 +/- 0.2 nm which decreases to 2.5 +/- 0.1 nm layer(-1) in the bulk, the reduction arising from an interdigitating layer arrangement, both top and bottom. It is the first example of LB-Lego(R) and, in addition, represents the only fully interdigitating structure with non-centrosymmetrically aligned chromophores. They are tilted 38 degrees from the substrate normal. The second-harmonic intensity increases quadratically with the number of layers, i.e. as I-(N)(2 omega) = (I(1)N2)-N-2 omega, with a second-order susceptibility of chi ((2))(zzz) = 30 pm V-1 at 1064 nm for refractive indices of n(omega) = 1.55 and n(2 omega) = 1.73, d = 2.5 nm layer(-1) and phi = 38 degrees. Angle resolved X-ray photoelectron spectra (XPS) of these films provide no evidence of the bromide counterion, which suggests that it is replaced by OH 2 or HCO3-, which occur naturally in the aqueous subphase, or C21H43COO- from the co-deposited fatty acid. This probably applies to all cationic dyes deposited by the LB technique.

Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation

Analogues of the potent, conformationally biased, decapeptide agonist of human C5a anaphylatoxin, C5a(65-74)Y65,F67,P69,P71,D-Ala73 (YSFKPMPLaR, peptide 54), were synthesized with methyl groups occupying specific C5a,, amide nitrogen atoms along the peptide backbone. This N-methylation induced crucial extended backbone conformations in a manner similar to the two Pro residues, but without eliminating the contributions made by the side-chain of the residue for which Pro was substituted. The presence of backbone N-methyl groups on peptide 54 analogues had pronounced detrimental effects on the ability to bind and activate C5aRs expressed on human PMNs, but not on the ability to contract smooth muscle of human umbilical artery. Several N-methylated analogues of peptide 54 (peptides 56, 67, 124, 125, and 137) were significantly more selective for smooth muscle contraction, which is mediated by tissue resident macrophages, than for enzyme release from PMNs. Indeed, peptide 67, YSFKDMP(MeL)aR was almost 3000-fold more selective for smooth muscle contraction than for PMN enzyme release. Consistent with these differential activities was the observation that peptide 67 expressed a significantly greater binding affinity to C5aRs expressed on rat macrophages than on rat PMNs. This differential activity was also observed in vivo in the rat where peptide 67 induced a hypotensive response similar to peptide 54 and rhuC5a, but without accompanying neutropenia. (C) 2001 Elsevier Science B.V. All rights reserved.

Total synthesis and absolute stereochemistry of plakortone D

The first total synthesis of plakortone D is described and thereby establishes the structure and absolute stereochemistry of the most biologically active member of the marine-derived plakortone family. The sterically congested bicyclic lactone core results from a Pd(II)-induced hydroxycyclization−carbonylation−lactonization sequence on an enediol whose chirality was installed by AD-technology. Attachment of the side chain, also constructed using AD-methodology, was achieved by using a modified Julia coupling. The described approach enables acquisition of other plakortones and analogues, in the correct (natural) stereochemical series.

Crystal structures of free, IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 Angstrom resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5-monophosphate (GMP) (2.8 Angstrom resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 Angstrom. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, Usually Mg2+ in vivo. In the free structure, a Mg2+, is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp 163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg2+ complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.

The radiation chemistry of ultem at 77 K as revealed by ESR spectroscopy

Radical formation in ultem following gamma-radiolysis has been reassessed, and the G(R*) values at different temperatures have been determined by ESR spectroscopy. The radical assignment and radical reactivity have been re-examined by photobleaching and thermal annealing studies. Photobleachable radical anions were found to comprise approximate to40% of the total number of radicals formed on radiolysis at 77 K. Spectral subtraction methods, ESR spectral simulations, measurement of g-values and the hyperfine splitting constants were used to identify the other radical intermediates. The principal chain scission radicals are formed due to scission of the main-chain at (i) the ether linkage, (ii) the isopropylidene group and (iii) the imide ring in the main chain. The side chain methyl groups of the isopropylidine units also lose hydrogen to form methylene radicals. The five-line spectrum observed to decay in the temperature range 370-430 K, which has not been assigned previously, has been identified as being characteristic of a di-substituted benzyl radical. (C) 2002 Elsevier Science Ltd. All rights reserved.

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH2O), Thr (-CH3CHO) and Asp (-H2O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert. Copyright (C) 2002 John Wiley Sons, Ltd.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Bioactive albumin functionalized polylactic acid membranes for improved biocompatibility

Biocompatibility is a major challenge for successful application of many biomaterials. In this study the ability to coat chemically and enzymatically activated poly(L-lactic acid) (PLA) membranes with heat denatured human serum albumin to improve biocompatibility was investigated. PLA membranes hydrolyzed with NaOH or cutinase and then treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, hydrochloride (EDAC) as a heterobifunctional cross-linker promoted the coupling single bondCOOH groups on PLA membranes and single bondNH2 groups of heat denatured human serum albumin. This resulted in increased hydrophilicity (lowest water contact angles of 43° and 35°) and highest antioxidant activity (quenching of 79 μM and 115 μM tetramethylazobisquinone (TMAMQ) for NaOH and cutinase pretreated membranes, respectively). FTIR analysis of modified PLA membranes showed new peaks attributed to human serum albumin (amide bond, NH2 and side chain stretching) appearing within 3600–3000 cm−1 and 1700–1500 cm−1 (Fig. 3). MTT studies also showed that osteoblasts-like and MC-3T3-E1 cells viability increased 2.4 times as compared to untreated PLA membranes. The study therefore shows that this strategy of modifying the surfaces of PLA polymers could significantly improve biocompatibility.

Estudo das propriedades dos adjuvantes na compatibilidade/robustez cimento/adjuvante

A qualidade do betão pode ser controlada pelo comportamento da fluidez da pasta de cimento, o qual está relacionado com a dispersão das partículas de cimento. Um dos maiores avanços na tecnologia do betão tem sido o desenvolvimento de aditivos. Um destes tipos de aditivos, os Superplastificantes (SP), fornecem a possibilidade de se obter uma melhor dispersão das partículas de cimento, produzindo pastas com elevada fluidez. Com o desenvolvimento de betões de alta resistência e elevado desempenho, os superplastificantes tornaram-se indispensáveis. Os superplastificantes são adsorvidos nas partículas de cimento e esta adsorção depende da composição do clínquer do cimento e do tipo de SP utilizado. Com a difusão do emprego dos aditivos redutores de água, têm surgido vários problemas de compatibilidade cimento/adjuvante. Esta investigação dedicada aos superplastificantes, fortes redutores de água, visou estudar quais as propriedades que poderiam influenciar a sua compatibilidade/robustez com o cimento. Também se procurou ganhar experiência com as técnicas analíticas de caracterização de adjuvantes. Assim, utilizou-se um tipo de cimento e dois tipos de superplastificantes (poli(étercarboxilatos) e poli(naftalenossulfonatos)) disponíveis no mercado português. Mantendo a mesma razão água/cimento (A/C), pretendeu-se determinar a natureza química, grau de funcionalização, teor e tipo de contra-ião, teor de sulfatos/sulfonatos do adjuvante e o comportamento dos superplastificantes nas pastas cimentícias, de forma a poder determinar indicadores de compatibilidade entre cimentos e superplastificantes. Constatou-se que a natureza química, o grau de funcionalização e a quantidade consumida dos superplastificantes têm influência nas pastas. Os indicadores de compatibilidade por parte dos superplastificantes parecem estar relacionados com o comprimento da cadeia lateral de éter e com o rácio CO2R/CO2 -. A alteração do momento da adição do adjuvante tem influência na compatibilidade cimento/adjuvante, sendo benéfico para os poli(étercarboxilatos) e prejudicial para o poli(naftalenossulfonato).