Solution conformation of an oligonucleotide containing a G.G mismatch determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.     

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Goldfish cones secrete a two-repeat interphotoreceptor retinoid-binding protein

Vitamin A and fatty acids are critical to photoreceptor structure, function, and development. The transport of these nutrients between the pigment epithelium and neural retina is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP, a 133-kDa (human) glycolipoprotein, is the major protein component of the extracellular matrix separating these two cell layers. In amphibians and mammals, IRBP consists of four homologous repeats of about 300 amino acids which form two retinol and four fatty acid-binding sites. Here we show that IRBP in teleosts is a simpler protein composed of only two repeats. Western blot analysis shows that goldfish IRBP is half the size (70 kDa) of IRBP in higher vertebrates. Metabolic labeling studies employing Brefeldin A taken together with in situ hybridization studies and the presence of a signal peptide show that goldfish IRBP is secreted by the cone photoreceptors. The translated amino acid sequence has a calculated molecular weight of 66.7 kDa. The primary structure consists of only two homologous repeats with a similarity score of 52.5%. The last repeats of human and goldfish IRBPs are 69.1% similar with hydrophobic regions being the most similar. These data suggest that two repeats were lost during the evolution of the ray-finned fish (Actinopterygii), or that the IRBP gene duplicated between the emergence of bony fish (Osteichthyes) and amphibians. Acquisition of a multirepeat structure may reflect evolutionary pressure to efficiently transport higher levels of hydrophobic molecules within a finite space. Quadruplication of an ancestral IRBP gene may have been an important event in the evolution of photoreceptors in higher vertebrates. Correspondence to: F. Gonzalez-Fernandez     

Homology modeling of histidine-containing phosphocarrier protein and eosinophil-derived neurotoxin: Construction of models and comparison with experiment

Homology modeling methods have been used to construct models of two proteins—the histidine-containing phosphocarrier protein (HPr) from Mycoplasma capricolum and human eosinophil-derived neurotoxin (EDN). Comparison of the models with the subsequently determined X-ray crystal structures indicates that the core regions of both proteins are reasonably well reproduced, although the template structures are closer to the X-ray structures in these regions—possible enhancements are discussed. The conformations of most of the side chains in the core of HPr are well reproduced in the modeled structure. As expected, the conformations of surface side chains in this protein differ significantly from the X-ray structure. The loop regions of EDN were incorrectly modeled—reasons for this and possible enhancements are discussed. © 1995 Wiley-Liss, Inc.     

Comparison of the bis-intercalating complexes formed between either ditercalinium or a flexible analogue and d(CpGpCpG)2 or d(TpTpCpGpCpGpApA)2 minihelices: 1H- and 31P-NMR analyses.

The 400-MHz 1H- and 162-MHz 31P-nmr have been used to study complexes constituted by (a) the d(TpTpCpGpCpGpApA)2 or the d(CpGpCpG)2 self-complementary oligonucleotides and (b) two bifunctional 7H-pyrido [4,3-c] carbazole dimer drugs, the antitumoral ditercalinium (NSC 366241), a dimer with a rigid bis-piperidine linking chain and its pharmacologically inactive analogue, a dimer with a flexible spermine-like linking chain. Nearly all proton and phosphorus signals have been assigned by two-dimensional (2D) nmr (correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser enhancement spectroscopy, 2D 31P (1H) heteronuclear correlated spectroscopy and 31P-31P chemical exchange experiments). Both drugs bis-intercalate into the two CpG sites. The complexes show small differences in the position of the 7H-pyrido [4,3-c] carbazole ring into the intercalation site and possibly in the ribose-phosphate backbone deformation. However, the inactive analogue exhibits a longer residence lifetime in octanucleotide than the ditercalinium does. All these results are discussed in terms of differences in dimer activities.     

Effect of 50 S subunit proteins L5, L18 and L25 on the fluorescence of 5 S RNA-bound ethidium bromide

Among the three Escherichia coli 50 S subunit proteins L5, L18 and L25, which have an affinity for 5 S RNA, only protein L18 exerts a strong effect on the fluorescence of 5 S RNA-ethidium bromide complexes, without changing the quantum yield of the fluorescence. Proteins L5 and L25, although they have little effect on the fluorescence, have a strong stabilizing influence on the 5 S RNA-L18 complex. The results are discussed in terms of the secondary and tertiary structures of 5 S RNA in relation to ribosomal protein binding.     

In Vivo versus Augmented Reality Exposure in the Treatment of Small Animal Phobia: A Randomized Controlled Trial

Although in vivo exposure is the treatment of choice for specific phobias, some acceptability problems have been associated with it. Virtual Reality exposure has been shown to be as effective as in vivo exposure, and it is widely accepted for the treatment of specific phobias, but only preliminary data are available in the literature about the efficacy of Augmented Reality. The purpose of the present study was to examine the efficacy and acceptance of two treatment conditions for specific phobias in which the exposure component was applied in different ways: In vivo exposure (N = 31) versus an Augmented Reality system (N = 32) in a randomized controlled trial. “One-session treatment” guidelines were followed. Participants in the Augmented Reality condition significantly improved on all the outcome measures at post-treatment and follow-ups. When the two treatment conditions were compared, some differences were found at post-treatment, favoring the participants who received in vivo exposure. However, these differences disappeared at the 3- and 6-month follow-ups. Regarding participants’ expectations and satisfaction with the treatment, very positive ratings were reported in both conditions. In addition, participants from in vivo exposure condition considered the treatment more useful for their problem whereas participants from Augmented Reality exposure considered the treatment less aversive. Results obtained in this study indicate that Augmented Reality exposure is an effective treatment for specific phobias and well accepted by the participants.     

Reassessment of structural characteristics of the d(CGCG)2:actinomycin D complex from complete 1H and 31P NMR

Complexes formed between Actinomycin D (ActD) and the tetranucleotides d(AGCT)2 and d(CGCG)2 were studied in detail by one and two-dimensional 1H and 31P NMR. The 31P two dimensional chemical exchange experiment, at room temperature on saturated complexes (1:1), showed unambiguously that the asymmetrical phenoxazone ring binds to the unique GC site under the two possible orientations in the d(AGCT)2 tetranucleotide but adopts a single orientation in the d(CGCG)2 tetranucleotide. For the d(CGCG)2:Act D saturated complex, complete assignments of all protons and phosphorus signals of the two-nucleotide strands, as well as of the two cyclic pentapeptide chains has allowed us to study in details the conformational features of the complex from NOE and coupling constants analysis. The tetranucleotide remains in a right-handed duplex, but the sugar puckers are modified for residues at the intercalation site. A uniform C2' endo pucker is observed for residues on the strand facing the quinoid side of the phenoxazone ring while a C2' endo-C3-endo equilibrium about 60% of C2' endo is proposed for the two residues on the strand facing the benzenoid side of the phenoxazone ring. In contrast to previous studies on ActD-DNA interactions, we have been able to measure the 3J phosphorus-proton coupling constants at the intercalation site but also adjacent to it, showing that 31P chemical shifts are not simply related to the backbone conformation. Molecular mechanics calculations, using empirical distances deduced from NOE effects as restrained distances during minimizations, led to a model differing mainly from those previously published by orientation of the N methyl groups of both N-Methyl-Valines.