Structural features of EDG1 receptor-ligand complexes revealed by computational modeling and mutagenesis.
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Amino Acid Sequence↗
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Cation-dependent structural features of beta-casein-(1-25).
Complete sequence-specific, proton-resonance assignments have been determined for the calcium phosphate-stabilizing tryptic peptide beta-casein-(1-25) containing the phosphorylated sequence motif Ser(P)(17)-Ser(P)-Ser(P)-Glu-Glu(21). Spectra of the peptide have been recorded, in separate experiments, in the presence of excess ammonium ions, sodium ions and calcium ions, and of the dephosphorylated peptide in the presence of excess sodium ions. We observed significant changes to chemical shifts for backbone and side-chain resonances that were dependent upon the nature of the cation present. Medium-range nuclear Overhauser effect (nOe) enhancements, characteristic of small structured regions in the peptide, were observed and also found to be cation dependent. The secondary structure of the peptide was characterized by sequential and medium-range (i, i+2/3/4, which denotes an interaction between residue i and residue i+2, i+3 or i+4 in the peptide) nOe connectivities, and Halpha chemical shifts. Four structured regions were identified in the calcium-bound peptide: residues Arg(1) to Glu(4) were involved in a loop-type structure, and residues Val(8) to Glu(11), Ser(P)(17) to Glu(20) and Glu(21) to Thr(24) were implicated in beta-turn conformations. Comparison of the patterns of medium-range nOe connectivities in beta-casein-(1-25) with those in alpha(S1)-casein-(59-79) suggest that the two peptides have distinctly different conformations in the presence of calcium ions, despite having a high degree of sequential and functional similarity.
Amino Acid Sequence↗
Structural features of angiotensin II which are important for biologic activity.
In summary, the aromatic ring of phenylalanine (COOH-terminus) is the key to the intrinsic activity with Tyr4, also involved in activation of a response. The NH2-terminal residue is a determinant of duration of action. The side chains of Tyr4 and His6 are key determinants for affinity, and the amino acids in the No. 1 and No. 2 position also contribute to receptor binding. Val3, Ile5, and Pro7 contain neutral side chains which probably establish and/or help maintain the appropriate distances among the key sidechains of Tyr4, His6, and Phe8. In other words, Val3, Ile5 (or Val), and Pro7 are key determinants of structural conformation.
Amino Acid Sequence↗
Induction of cytochrome P4501A (CYP1A) by clotrimazole, a non-planar aromatic compound. Computational studies on structural features of clotrimazole and related imidazole derivatives.
The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). High-affinity AhR ligands are planar polyaromatic molecules such as the prototypic ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The present work investigated the ability of the imidazole derivative, clotrimazole [1-(2'chlorotrityl)imidazole, CLO], to induce CYP1A in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes at the catalytic activity (determined as 7-ethoxyresorufin-O-deethylase, EROD) and at the transcriptional level. CLO resulted in a significant increase of hepatocyte EROD activity and CYP1A mRNA at a concentration of 1.56 microM. Computational studies on the molecular structure of CLO show that CLO is unlikely to take a planar conformation. Further indications that CLO does not behave like a planar AhR ligand come from the experimental observation that co-incubation of trout hepatocytes with CLO and the AhR antagonist, alpha-naphthoflavone (alpha-NF), did not result in an inhibition of CLO induction of CYP1A mRNA, whereas alpha-NF was able to inhibit CYP1A induction by the prototpyic, planar AhR ligand, beta-naphthoflavone. The experimental findings on CLO agree with previous results obtained for another non-planar imidazole derivative, 1-benzylimidazole (BIM). Further, computational studies showed that the non-planar imidazoles, BIM and CLO, are highly similar with respect to some electrostatic properties, namely the dipole moment and the molecular electrostatic potential (MEP). Overall our experimental and computational studies suggest that transcriptional activation of CYP1A by the imidazole derivatives CLO and BIM is mediated by a mechanism different to that of prototypic CYP1A inducers such as the planar AhR-ligands.
Animals↗
Unique structural features of red kidney bean purple acid phosphatase.
Purple acid phosphatase from red kidney beans (Phaseolus vulgaris) has been purified to homogeneity and characterized. The enzyme is a homodimer of 60 kDa subunits each containing one atom of zinc and iron in the active site. Circular dichroism spectral studies on the purified enzyme reveals that a large portion of the peptide backbone is in the unordered and beta-turn conformation. A unique feature of the red kidney bean acid phosphatase, which we have found, is that one of the two cysteines of each subunit is involved in the formation of an inter-subunit disulphide. The thiol group of the other cysteine is not necessary for the activity of the enzyme. Western blot analysis with antibodies raised against kidney bean acid phosphatase could not recognize acid phosphatases from other sources except from potato. This paper emphasizes the fact that acid phosphatases are functionally, but not structurally, conserved enzymes.
Acid Phosphatase↗
Study of structural features and thermodynamic parameters, determining the chromatographic behaviour of drug-cyclodextrin complexes.
The chromatographic behaviour of host-guest inclusion complexes was studied, in order to predict the optimal conditions for their accurate analysis and overcome the significant analytical errors generated by the presence of cyclodextrins. Complexes of tolfenamic acid and ketoprofen with beta-cyclodextrtin (betaCD), 2-hydroxypropyl-betaCD (HPbetaCD) and methyl-betaCD (MebetaCD) prepared in different molar ratios, were studied. Since the drug release from cyclodextrins' complexes is a prerequisite for its accurate quantitation, several parameters affecting the dissociation during the analysis were evaluated. In an attempt to explain the drug release mechanism from cyclodextrins, during HPLC analysis, the possible correlation of the NMR structural findings with the binding constants and the thermodynamic quantities of complexation were examined, in relation to their chromatographic behaviour. Finally, the presence of the solvation spheres around the supramolecules, which affect the complex stability, is suggested to be crucial for our chromatographic findings. Particularly, entropy change in the system is considered the most critical factor, determining the time required for dissociation of drug-cyclodextrin complexes, during drug quantitation.
Chromatography, High Pressure Liquid↗
Structural features facilitating the glutamate dehydrogenase catalyzed alpha-imino acid-alpha-amino acid interconversion.
Glutamate dehydrogenase catalyzes the reversible oxidation of L-proline and L-pipecolic acid to the corresponding cyclic alpha-imino acids. The active substrates are the amino acid anion in one direction and the iminium ion in the other. The oxidation of the ester, amide, and N-methyl derivatives of L-proline by enzyme-NADP+ and the reduction of N-methyl-delta 1-tetrahydropyridinium ion by enzyme-NADPH do not proceed to a detectable extent under the experimental conditions. The methyliminium ion, however, undergoes facile nonenzymatic reduction by NADPH. If it is assumed that the nonenzymatic reaction reflects the structural requirements of the redox step of the enzymatic reaction, then the lack of reactivity of the tetrahydropyridinium ion toward enzyme-NADPH must be due to the instability of the central complex. It appears that the alpha-carboxylate and NH groups in the amino acid anion and in the alpha-imino acid are involved in binding the substrates to the enzyme-coenzyme complexes. We conclude that each of these active substrates binds to its appropriate enzyme-coenzyme complex through a hydrogen bond between its NH group and a basic enzyme group; there is also an ionic bond in the central complex between the alpha-carboxylate group of the active substrate and a positively charged enzyme group. The five-membered amino acid anions are more reactive toward enzyme-NADP+ than the six-membered ones. The same reactivity order is seen for the reduction of imino acids by enzyme-NADPH. Since these effects are also present in the nonenzymatic reduction by NADPH we ascribe the ring size effects on V/Ksubstrate primarily to those on the hydride-transfer step.
Amino Acids↗
Pancreatic procarboxypeptidases: their activation processes related to the structural features of the zymogens and activation segments.
The molecular events leading to the complete activation of pancreatic procarboxypeptidases A and B have been investigated. For both proteins the activation process follows a similar general scheme: trypsin is responsible for the first cleavage that separates the active enzyme from the activation segment, the degradation of the activation segment proceeds only from its C-terminal end, and activity release can be correlated with the disappearance of the long forms of the activation segment. In both systems, trypsin and the released carboxypeptidase participate in the trimming of the severed activation regions. However, the rate of enzymatic activation is much faster in the case of procarboxypeptidase B. This phenomenon may be explained by some structural differences in the connecting region which acts as a linker between the globular domain of the activation segment and the N-terminal end of carboxypeptidases and also by the higher efficiency of carboxypeptidase B for the digestion of its own activation segment. It is not due to unfolding of the activation domain, since the isolated activation domain retains its globular conformation in solution.
Amino Acid Sequence↗
Sterically stabilized BGTC-based lipoplexes: structural features and gene transfection into the mouse airways in vivo.
BACKGROUND: Colloidal stability of lipid/DNA aggregates is a major requirement for cationic lipid-mediated transfection which is particularly difficult to fulfil at the high DNA concentrations used for in vivo gene delivery. Thus, we have investigated the potential of poly(ethyleneglycol) (PEG) conjugates for steric stabilization of lipoplexes formed by bis(guanidinium)-tren-cholesterol/dioleoyl phosphatidylethanolamine (BGTC/DOPE) liposomes, a class of cationic liposomes we have developed over the past few years. METHODS AND RESULTS: We demonstrate that adequate lipophilic PEG derivatives can stabilize BGTC/DOPE lipoplexes formed at high DNA concentration. We also report the results of cryotransmission electron microscopy studies indicating that PEG-stabilized lipoplexes form DNA-coated structures which assemble into clusters exhibiting various complex morphologies. Finally, we report data from in vivo transfection experiments suggesting that PEG-mediated colloidal stabilization of concentrated lipoplex solutions may allow enhanced transfection of the mouse airways via intranasal administration. CONCLUSION: Our results represent an important step towards the design of multimodular BGTC-based systems for improved in vivo gene transfection.
Animals↗
Common structural features of the genes for two stable RNAs from Halobacterium halobium.
The genes coding for the 5S rRNA and another stable RNA, termed 7S RNA, in Halobacterium halobium were isolated from a genomic library of this archaebacterium and their nucleotide sequences determined. Both genes are colinear with their transcripts (5S rRNA and 7S RNA), but 5S rRNA and possibly also 7S RNA isolated from other halobacteria carry additional nucleotides within the RNA transcript. Both genes are located in the G + C rich chromosomal fraction I of H. halobium. Comparison of the 3' non-coding regions of both genes shows a 20 bp sequence of high homology immediately at the 3' ends which is almost symmetrically flanked by two stem-loop structures, one being situated close to the 3' end but within the coding region and the other downstream of the common 20 bp sequence.
Base Sequence↗
Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues.
We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.
Animals↗
New structural features of synapses in the anteroventral cochlear nucleus prepared by direct freezing and freeze-substitution.
The rostral anteroventral cochlear nucleus (AVCN) of the chinchilla provides a preparation in which neuronal cell bodies and synapses in the mammalian central nervous system can be examined after direct freezing and freeze-substitution of rapidly excised brain stem slices. Cell bodies and synapses in the freeze-substituted AVCN differed from those in perfusion-fixed AVCN in several interesting respects. Despite of these differences, four types of synaptic terminal were distinguished in freeze-substituted AVCN and correlated with the four well-known types of perfusion-fixed terminal. Since the transmitter at each of the four types of terminal has been tentatively identified, the structure of synaptic vesicles and junctions in the freeze-substituted terminals could be related to transmitter type. Synaptic vesicles were uniformly round, but their diameters, deployment, and related cytoskeletal elements near the synaptic junction differed in each chemical type of synapse; the synapses thought to be cholinergic, for instance, had only a few vesicles clustered at their presynaptic junctions while the rest of the vesicles were separated from the junction by a network of fine filaments. Two types of filamentous components, short vertical projections from the postsynaptic membrane and thin filaments protruding from these projections, comprised the basic structure of the postsynaptic specialization, but their sizes and distribution differed at each chemical type of terminal. For instance, the postsynaptic specialization at the glycine terminal was distinguished by numerous thin filaments which curved sideways to run parallel to the plasmalemma. Thus, freeze-substitution gives new information about structural differences between chemically different types of synapses, which may reflect differences in their transmitter storage, release, and reception. In addition, the AVCN preparation is of general interest in making cell bodies in the mammalian central nervous system available to the various structural and analytical techniques which depend on direct, rapid freezing.
Animals↗
Structural features of fungal beta-D-glucans for the efficient inhibition of the initiation of virus infection on Nicotiana tabacum.
Glucans of fungal origin have been shown to inhibit the early stages of infection of Nicotiana by numerous viruses of different taxonomic groups. Several glucans were isolated from the cell walls of Phytophthora parasitica, Phytophthora megasperma f. sp. glycinea (Pmg) and Fusarium oxysporum, and their antiviral activity compared on tobacco leaves inoculated with tobacco mosaic virus. These polysaccharides consist of a mixture of (1-->3)(1-->6)-beta-D-glucans with M(r) varying from 1.1 x 10(3) to 2 x 10(6). Requirements for a prominent antiviral activity of the fungal polysaccharides are a beta-(1-->3)(1-->6)-D-glucan structure with mono-, di-, tri- or tetra-glucosidic side branches attached to a linear main chain of beta-(1-->3)-linked-D-glucose residues. Very high activity is correlated with a high degree of branching at position 6 and with the size and glycosidic nature of the side chains. The molecular masses and the organized structure of fungal beta-D-glucans are not essential for their antiviral activity. The structural motif for antiviral activity in Nicotiana is distinct from that required for elicitation of phytoalexins in soybean cotyledons.
Antiviral Agents↗
Proton NMR and structural features of a 24-nucleotide RNA hairpin.
The three-dimensional conformation of a 24-nucleotide variant of the RNA binding sequence for the coat protein of bacteriophage R17 has been analyzed using NMR, molecular dynamics, and energy minimization. The imino proton spectrum is consistent with base pairing requirements for coat protein binding known from biochemical studies. All 185 of the nonexchangeable protons were assigned using a variety of homonuclear 2D and 3D NMR methods. Measurements of nuclear Overhauser enhancements and two-quantum correlations were made at 500 MHz. New procedures were developed to characterize as many resonances as possible, including deconvolution and path analysis methods. An average of 21 distance constraints per residue were used in molecular dynamics calculations to obtain preliminary folded structures for residues 3-21. The unpaired A8 residue is stacked in the stem, and the entire region from G7 to C15 in the upper stem and loop appears to be flexible. Several of these residues have a large fraction of S-puckered ribose rings, rather than the N-forms characteristic of RNA duplexes. There is considerable variation in the low-energy loop conformations that satisfy the distance constraints at this preliminary level of refinement. The Shine-Dalgarno ribosome binding site is exposed, and only two apparently weak base pairs would have to break for the 16S ribosomal RNA to bind and the ribosome to initiate translation of the replicase gene. Although the loop form must be regarded as tentative, the known interaction sites with the coat protein are easily accessible from the major groove side of the loop.
Base Sequence↗
Structural features of phi29 single-stranded DNA-binding protein. II. Global conformation of phi29 single-stranded DNA-binding protein and the effects of complex formation on the protein and the single-stranded DNA.
The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.
Bacillus Phages↗
Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs.
The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t(6)A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) has a weaker positive effect depending on the context of other modifications. The s(2)U34 modification apparently has none and the pseudouridine (psi39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp(287) contacts mnm(5)s(2)U34 was reinforced by the observations that the mammalian tRNA(Lys-3) wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm(5)s(2)U) is inhibitory and that the D287H mutant favors tRNA(Lys-3) over Escherichia coli tRNA(Lys). The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA(Lys-3) primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.
Kinetics↗
DNA adducts with chlorophyll and chlorophyllin as antimutagenic agents: synthesis, stability, and structural features.
Porphyrins and their metal derivatives are strong DNA binders with association constants of 10(5) M(-1) to 10(7) M(-1). Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. Chlorophyll (CHL) and chlorophyllin (CHLN), a food-grade derivative of chlorophyll, the ubiquitous green plant pigment widely consumed by humans, are potent inhibitors of experimental carcinogenesis. The aim of this report was to examine the interaction of calf-thymus DNA with CHL and CHLN in aqueous solution at physiological pH, with pigment/DNA(phosphate) molar ratios (r) of 1/80 to 1/2. Fourier transform infrared (FTIR) difference spectroscopic method was used to determine the pigment binding mode, binding constant, sequence selectivity, DNA secondary structure and structural variations of the pigment-DNA complexes in aqueous solution.
Animals↗
Retinol and retinoic acid bind human serum albumin: stability and structural features.
Vitamin A components, retinol and retinoic acid, are fat-soluble micronutrients and critical for many biological processes, including vision, reproduction, growth, and regulation of cell proliferation and differentiation. The cellular uptake of Vitamin A is through specific interaction of a plasma membrane receptor with serum retinol-binding protein. Human serum albumin (HSA), as a transport protein, is the major target of several micronutrients in vivo. The aim of present study was to examine the interaction of retinol and retinoic acid with human serum albumin in aqueous solution at physiological conditions using constant protein concentration and various retinoid contents. FTIR, UV-vis, CD and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant and the effects of complexation on protein secondary structure. Structural analysis showed that retinol and retinoic acid bind non-specifically (H-bonding) via protein polar groups with binding constants of K(ret)=1.32 (+/-0.30)x10(5)M(-1) and K(retac)=3.33 (+/-0.35)x10(5)M(-1). The protein secondary structure showed no alterations at low retinoid concentrations (0.125 mM), whereas at high retinoid content (1mM), an increase of alpha-helix from 55% (free HSA) to 60% and a decrease of beta-sheet from 22% (free HSA) to 18% occurred in the retinoid-HSA complexes. The results point to a partial stabilization of protein secondary structure at high retinoid content.
Humans↗