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The X-ray crystal structure of thioredoxin reductase from Drosophila melanogaster (DmTR) at 2.4-Angstrom resolution in which the enzyme was truncated to remove the C-terminal tripeptide sequence Cys-Cys-Ser is presented. Results reveal that DmTR has a more open tetrapeptide binding pocket than the mouse enzyme and accommodates the peptide Ser-Cys-Cys-Ser(ox) in a cis conformation.

The study was conducted for the cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) as the center of organization of the red blood cell as well as several metabolic enzymes and hemoglobin. In conclusion the data provides the first demonstration of a defined change in the molecular structure of cdAE1, and also indicates that the structure under physiological conditions is different from the crystal structure determined at low pH.

Point mutational study reveals that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit contribure a 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Further, residues Lys-34, Gly-57, Asp-59 and Asp-173 contribute to the high affinity of the alpha-epsilon site than that of alpha-delta site.

A study of nucleotides at the corner of the L-shaped tRNA has been conducted to demonstrate that each of them contribute to the editing efficiency. Results indicate that these nucleotides translocate amino acids from the active side to the center for editing.

Research was conducted to study the influence of galanin or galanin-derived peptides on cAMP production, MAPK activity and the accumulation of inositol phosphate in the Chinese hamster ovary cell lines expressing the rat galanin receptor subtypes. Cells were split into 12-well plates and incubated for one to two days while DNA and protein sequence studies were carried out using the DNA* software. The physiological functions showed by galanin indicated the involvement of multiple galanin receptors activating distinct intracellular signaling pathways.

Freeze-quench electron paramagnetic resonance studies were carried out in the investigation of the enzyme kinetics of quinol reductase cytochrome bo3 from Escherichia coli under conditions of substrate limitation. This method allowed the researcher to accurately measure the potential of the heme b prosthesis and relate this to the electron transfer mechanics. Results indicate that proton translocation takes place by a Q-cycle mechanism.

A study was conducted on the effects of the (+)-trans-anti-BP-N2-dG lesion on DNA replication as well as the conformational features and interactions at a primer-template junction in the presence of a DNA polemerase. The results indicate that the benzo[a]pyrenyl moiety's main harmful impact on DNA synthesis may result in part from its specific interactions with key polymerase side chains.

Research was conducted to study the structural basis of mutagenesis influenced by the chemical carcinogens 2-aminofluorene and 2-acetylaminofluorene at a mutational hot spot sequence in DNA. Monoadducted dG isomers were transformed into the monoadducted [AF]dG isomers while the adduct duplex was collected with a jump-return pulse. Results showed that the [AF]dG5 adduct in an anti orientation about the glycosidic bond aligned through Watson-Crick pairing.

Research was conducted to study the conformation equilibrium between aminofluorene (AF)-intercalated and AF-external states in slow exchange for the (AF)dG lesion located at the opposite dC in the sequence context. A molecular mechanics program was utilized to determine minimized potential energy calculations. Results suggested that population shifts may denote a much larger size of the pyrenyl ring of the (AP)dG adduct compared to the fluorenyl ring of the (AF)dG adduct.

It is important to outline the structural properties of (AF)dG adduct to be able to determine the relationship to mutagenesis. The structure of the Csuper8-(AF)dG adduct is addressed, to ascertain if there are common structural features between the pairs of adducts. There were differences in the covalent linkage site for the Csuper8-(AF)dG and (+)-trans-anti-Nsuper2-(BP)dG adducts. The results support earlier proposals that the adduct alignment could represent a common mutagenic conformer.

NMR methods have have used to provide insights into the structure of (AF)dG adducts. Structural issues related to the capacity of AF fro frameshift mutations of the -2 type on covalent adduct formation at the Gsuper3 site in the NarI sequence context, are addressed. NMR-computational structural studies concentrated on a 12/10-mer duplex, and the research emphasised the formation of just one of two possible AF-intercalation alignments of (AF)dG in the NarI sequence.

The links between polycyclic aromatic hydrocarbon (PAH) carcinogen structures are of great interest to chemists and biologists. Benzo(a)pyrene (BP) is a PAH compound, and both (+)- and (-)-anti-BPDE undergo a chemical reaction with DNA by binding mainly to exocyclic amino groups. The conformations of four (BP)dG lesions located opposite dC at the 11-mer DNA duplex level, have been determined, using NMR techniques.

The solution conformation of the covalent (+)-trans-anti-(BP)dG adduct is reported, located opposite dC at the junctional site in the 13/10-mer DNA sequence. 13-mer acts as a template strand, incorporating the junction (BP)dG4 lesion. Intramolelcular and intermolecular proton-proton distances were defined by lower and upper bounds determined from NOESY spectra.

The structure of N-(deoxyguanosine-8-yl)-1-aminopyrene ([AP]dG) 11-mer duplex was analyzed by nuclear magnetic resonance. The C8 deoxyguanosine adduct exhibited a structural alignment composed of aminopyrene rings that are covalently attached to the duplex. The aminopyrene rings are intercalated with two intact base pairs of Watson-Crick dC5.dG18 and dC7.dG1 with a tilted orientation that forms novel nuclear Overhauser effects.

The covalently attached Benzo(a)pyrenyl ring (BP) intercalates between the dC5.dG18 and dC7.dG16 base pairs of the DNA duplex molecule and displaces deoxyguanosine (BP)-dG6 and its partner cytosine dC17 from the helix into the major groove. The benzylic ring in the intercalated BP ring is oriented towards the minor groove in the (+)cis-anti-(BP)dG.dC duplex isomer and towards the major groove in the (-)cis-anti isomer. Both duplex adducts displace the modified deoxyguanosine into the major groove.

The aminofluorene ring of aminofluorene-C8-deoxyguanosine ({AF}dG) is intercalated between the Watson-Crick dC5.dG18 and dC8.dG17 base pairs. The deoxyguanosine base of (AF)dG, positioned in the major groove, shows a syn alignment. The solution conformation of (AF)dG adduct opposite a -2 deletion site indicates the involvement of stabilizing interactions. The base displacement and carcinogen-base stacking are comparable to the solution conformation of (AF)dG adduct opposite a -1 deletion site.

The structural characteristics of (+) and (-)-trans-anti-benzo(a)-pyrene-dG adducts located at the junction site of single-strand/duplex 13/9-mer sequence were determined. The analyses involve the use of MNR spectrometry in combination with molecular mechanics calculations. The results showed that the modified 13-mer strand mimics the template strand, while the complementary 9-mer strand mimics a primer which extends from the 3'-end of the template toward the 5'-end up to the base preceding the modified guanine.

Two-dimensional nuclear magnetic resonance molecular mechanics studies were conducted on the (-)-trans-anti-[MC]dG adduct 1 positioned opposite dC in the same DNA sequence 2 used in three previous structural studies of three stereospecific benzo[a]pyrenyl [BP]dG duplex adducts. Results revealed several similarities between the structures of the (-)-trans-anti-[MC]dGdC 11-mer duplex and analogous benzo[a]-pyrenyl(-)-trans-anti-[BP]dGdC 11-mer duplex. In addition, DNA bending was linked with wedging of the methyl group of 5-methylchrysene to the 3'-side of the modification site.

A study was conducted on the solution structure of [AF]dG adducts located opposite -1 deletion sites. A solution structural characterization of [AF]dG adduct 1 located opposite a deletion site in the sequence context d(C5-[AF]G6-C7)d(G16-G17) 2 at the DNA oligonucleotide duplex level was provided. Results showed that the hydrophobic aromatic rings of the heterocyclic aromatic amine and the polycyclic aromatic hydrocarbon preferentially intercalate into the helix opposite -1 deletion sites and bundle with flanking base pairs when covalently linked to guanines.

Examination of the solution structures of the (-)-trans-anti-benzo(c)phenanthrene-dA((BPh)dA) adduct opposite dT in a DNA duplex by a combined two-dimensional NMR-molecular mechanics approach reveals intercalation of the covalently attached benzo(c)phenanthrenyl ring with the 3'-side of the (-)-trans-anti-(BPh)dA adduct site. A comparative study reveals that the direction of this intercalation is opposite to that observed in the solution structure of the (+)-trans-anti-(BPh)dA opposite dT stereiosomers.

NMR and molecular mechanics studies of the solution structure of (+)-trans-anti-(BP)dG adduct present opposite the deletion site in DNA oligomer duplex reveal the occurrence of benzo(a)pyrene ring intercalation into the base pair-displaced helix of the (BP)dG6 deoxyguanosine base and displays numerous shifted proton resonances. The intercalation site is wedge-shaped but narrows in the dG16-dG17 position of the deletion-containing strand. Solution structure of the intercalated 11-mer duplex does not correlate with the previously proposed structure where the pyrene ring localizes to the minor groove and does not affect the Watson-Crick base pairing.

Use of molecular mechanics and NMR to analyze the solution structure of (+)-cis-anti-(BP)dG adduct present opposite the deletion site of a DNA duplex show that the benzo(a)pyrene ring intercalates into the helix that is present opposite the deletion site, while the altered deoxyguanosine base undergoes displacement to the minor groove. The intercalation site has a wedge shape while the benzo(a)pyrene ring stacks over the Watson-Crick dG.dC base pairs. However, during the intercalation of (+)-trans-anti-(BP)dG, the modified base displaces into the major groove unlike the minor groove displacement of the base during (+)-cis-anti(BP)dG intercalation.

Two dimensional NMR methods, together with energy minimization computations, were used to examine the solution structure of (+)-trans-anti-(BPh)dA adduct located opposite the dT in the sequence context d(C5-(BPh)A6-C7)-d-(G16-T17-G18) at the 11-mer duplex level. The (BPh)dA dT base pair was not terminated when the covalently bound benzo (c) phenanthrenyl intercalated to the 5'-part of the (Bph)dA adduct region.

The structure of the (+)-cis-anti(BP)dG adduct designated opposite dC in solution with a DNA oligomer duplex was analyzed usin NMR-energy minimization calculations. Results indicated intercalation between specific Watson-Crick base pairs and the benzopyrene ring of (BP)dG6 in a right handed helix. Shifted protein resonances was observed in the intercalative structure because of ring current effects of the deoxyguanosine and pyrenyl rings of the adduct. These data showed a base-displayed intercalation type form for a polycyclic aromatic hydrocarbon fixed to the deoxyguanosine adduct at the DNA oligomer level.

Nuclear magnetic resonance spectroscopy was used to study the structure of the N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex d(C1-C2-A3-C4- AAF-G5 -C6-A7-C8-C9 d G10-G11-T12-G13-C14-G15-T16-G17-G18). Results revealed that eight of the complementary nucleotides take the form of Watson-Crick base pairs. However, no evidence was found to support the possibility that AAF-G5, which follows a syn conformation, and C14 bases form a complementary hydrogen bond to each other. It was also found that the remaining bases adopted an anti conformation.

A study was conducted on the effect of the metabolic activation of benzo(a)pyrene with 7,8-diol 9,10-epoxide derivatives on the solution conformations of DNA covalent adducts. Orientational differences were observed for the benzo(a)pyrene ring, and these were attributed to the chiral properties of the two benzo(a)pyrene diol epoxide enantiomers. The results suggest significant implications on the interaction of cellular enzyme systems with the two structurally different alignments oberved.

Molecular dynamics simulations followed by binding energy analysis (MM-GBSA method) were performed to demonstrate the structural and energetic features of an atomic-level model of a T20-gp41 complex embedded in an explicit DOPC membrane that contribute to drug resistance. The results from the study provide insight into the possible design of the peptide to increase the level of favorable contact with both the membrane and gp41 which would lead to enhanced activity.

The significant role of alpha4 which is a PP2Ac-interacting adaptor protein that provides a binding platform for both protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac) and ubiquitin ligase Mid1 is described. The results offer insight into the polyubiquitination of PP2Ac which is inhibited by the UIM within alpha4 and the direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.

The studies have shown that Cys- and selenocysteine (Sec)-containing thioredoxin reductases (TRs) are distinguished by the importance each class of enzymes places on the eight-membered ring structure in the catalytic cycle. The biochemical properties of the truncated Cys and Sec TR enzymes have shown that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH.

The investigation report of the C-terminal redox-active tetrapeptides of three different thioredoxin reductases (TRs) which was carried out by employing protein engineering techniques is presented.

The molecular basis for xeroderma pigmentosum complementation group C (XPC) function and mutational defects associated with XP disease is analyzed with the help of a series of stable bacterially expressed N- and C-terminal fragment. A structural model of a folded XPC core has offered key insights into how domains from the two portions of the protein might cooperate in generating specific XPC functions.

The ability of alkylating electrophiles to inhibit protein phosphatases is examined, applying model electrophile probes that exhibit chemistries relevant to diverse biological reactive intermediates. The identification of specific sites linked to protein phosphatase 2A (PP2A) inactivation suggests that specific assays for abducted forms of these sequences could provide a general means of screening PP2A inhibitors.

The synthesis of the (super 13)C-labeled substrate-analogue inhibitor TU-514 and the subsequent refinement of the solution structure of the Aquifex aeolicus LpxC-TU-514 complex using residual polar couplings are reported. A revaluation of the catalytic role of an active site histidine, H253, on the basis of both its pK(sub a) as determined by NMR titration and pH-dependent kinetic analyses is discussed.

The mechanism of acetyllysine hydrolysis by the histone deacetylase (HDAC) and the substrate specification of the HDAC enzymes and the kinetics of deacetylation catalyzed by intact HDAC-containing corepressor complexes are explored. The results are explained in terms of the biological roles of the HDAC enzymes and the proposed reaction mechanism of acettyllysine hydrolysis by these enzymes.

An attempt is made to investigate the structural properties of human mitochondrial tRNA (super Leu(UUR) along with a focus on domains within this molecule that appear unstable. Further, a series of tRNAs featuring mutations within the D and anticodon stems were prepared using nuclease probing.

Research describes a combined nuclear magnetic resonance-molecular mechanics computational approach for elucidating a structural view of the type of distortions induced into DNA duplex by alkylating agent omicron(sup)6-[4-oxo-4-(3-pyridyl)butyl]guanine, at the omicron(sup)6 position of guanine. Data show that the alkylating agent causes helical distortions, wobble base pairing, and melting temperature in the DNA.

Results show that total editing, the sum of pre- and post-transfer editing, by class I aminoacyl-tRNA synthetases involves both aminoacylation and deacylation activities. Data suggest a post-transfer deacylation event wih the formation of an editing-active enzyme/tRNA complex, which edits misactivated valyl-adenylate.