Solution structure of the minor conformer of a DNA duplex containing a dG mismatch opposite a benzo[a]pyrene diol epoxide/dA adduct: glycosidic rotation from syn to anti at the modified deoxyadenosine

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental contaminants whose metabolism in mammals results in deleterious cell transformation. Covalent modification of DNA by diol epoxides metabolically formed from PAHs such a benzo[a]pyrene (BaP) provides a mechanism for the genotoxicity, mutagenicity, and carcinogenicity of PAHs. We had previously reported NMR evidence for a minor conformer of the duplex d(G1G2T3C4A5*C6G7A8G9).d(C10T11C12G13G14G15A16C17C18) containing a dG14 mismatch opposite a dA5* residue modified at the exocyclic amino group by trans addition to (+)-(7R,8S,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene [Yeh, H.J.C., Sayer, J.M., Liu, X., Altieri, A.S., Byrd, R.A., Lashman, M.K., Yagi, H., Schurer, E.J., Gorenstein, D.G., & Jerina, D.M. (1995) Biochemistry 34, 13570-13581]. In the present work, we describe the structure of this minor conformer (ca. 17% of the total conformer population). This represents the first structural determination of a minor conformer of a carcinogen-lesion DNA adduct. Two-dimensional NOESY, ROESY, TOCSY, and exchange-only spectra at 750 MHz allowed nearly complete sequential assignment of both conformers. In the minor conformer, the adducted base assumes an anti-glycosidic torsion angle whereas in the major conformer it assumes an unusual syn-glycosidic torsion angle. The aromatic hydrocarbon in the minor conformer is intercalated between dG13 and dG14, preserving the energetically favorable stacking interactions found in the major conformer. The major structural differences between the two conformers appear to be near the lesion site as evidenced by the large chemical shift differences between major and minor conformer protons near the lesion site; away from this site, the chemical shifts of the major and minor conformer protons are nearly identical. Because any of the conformations of benzo[a]pyrene diol epoxide-modified DNA may contribute to tumorigenic activity, structural determination of all conformations is essential for the elucidation of the mechanism of cell transformation initiated by covalent modification of DNA by PAHs.     

Solution studies of isepamicin and conformational comparisons between isepamicin and butirosin A when bound to an aminoglycoside 6'-N-acetyltransferase determined by NMR spectroscopy

NMR spectroscopy, combined with molecular modeling, was used to determine the conformations of isepamicin and butirosin A in the active site of aminoglycoside 6'-N-acetyltransferase-Ii [AAC-(6')-Ii]. The results suggest two enzyme-bound conformers for isepamicin and one for butirosin A. The dihedral angles that describe the glycosidic linkage between the A and B rings for the two conformers of AAC(6')-Ii-bound isepamicin were phi AB = -7.9 +/- 2.0 degrees and psi AB = -46.2 +/- 0.6 degrees for conformer 1 and phi AB = -69.4 +/- 2.0 degrees and psi AB = -57.7 +/- 0.5 degrees for conformer 2. Unrestrained molecular dynamics calculations showed that these distinct conformers are capable of interconversion at 300 K. When superimposed at the 2-deoxystreptamine ring, one enzyme-bound conformer of isepamicin (conformer 1) places the reactive 6' nitrogen in a similar position as that of butirosin A. Conformer 2 of AAC(6')-Ii-bound isepamicin may represent an unproductive binding mode. Unproductive binding modes (to aminoglycoside modifying enzymes) could provide one reason isepamicin remains one of the more effective aminoglycoside antibiotics. The enzyme-bound conformation of butirosin A yielded an orthogonal arrangement of the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings, as opposed to the parallel arrangement which was observed for this aminoglycoside in the active site of an aminoglycoside 3'-O-phosphotransferase [Cox, J. R., and Serpersu, E. H. (1997) Biochemistry 36, 2353-2359]. The complete proton and carbon NMR assignments of the aminoglycoside antibiotic isepamicin at pH 6.8 as well as the pKa values for it's amino groups are also reported.     

Conformational change due to esterification of hydroxy groups in erythromycin A and its major metabolite: analysis of these derivatives with different biological properties using NMR and molecular dynamics (MD) data

A conformational study is performed on the acylated erythromycin and erythralosamine derivatives from comparison between experimental results (NMR) and theoretical calculations by Molecular Dynamics (MD) in attempts to correlate their conformations with their abilities to generate cytochrome P450-nitroso metabolite complexes in vitro. As the 3'-dimethyl-amino function of the desosamine is metabolized and responsible for the interaction with cytochrome P450, its position, mobility and steric hindrance in the proximity of this functional group are related to its biological properties. The major conformations of the lactone ring were termed A (A1, A2, A3) and B (B1, B2), and this macrocycle flexibility induced five different orientations a, b, c, d and e for the desosamine sugar. Conformations A and B differ in many ways but the major change is the inward folding of the C(3) fragment in B. Conformer a exhibits an orientation of the desosamine nearly perpendicular to the macrocycle whereas the two units are in the same plane in conformations c and e. For conformation b, the cladinose unit lifts up above the macrocycle. Conformation d exhibits a turned-back cladinose. In the erythromycin derivatives esterification at the beta position to the N(CH3)2 group of the desosamine reduces the degree of freedom of the macrocyclic lactone ring which corresponds to conformation A only. The desosamine sugar was found to be perpendicular to the macrocycle (a conformer) and both sugar groups are parallel to reduce the steric energy. In the erythralosamine derivatives, the macrocycle is always present as conformation B with the two conformations b and c of the sugar rings. The steric parameters favour the b conformers in which the amino group is tilted up, while in 3,2'-dibenzoylated stacking aromatic attraction stabilizes the planar c conformer. Both isomers are thus shown to adopt well-defined conformations and to be well-adapted for a comparative structure-activity correlation studies. There is a significant relationship between the conformation b and the formation of cytochrome P450-nitroso metabolite complexes.     

Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.     

Dynamics of protein-bound water in the heme domain of P450BM3 studied by high-pressure spectroscopy: comparison with P450cam and P450 2B4

Pressure-induced transitions in the heme domain of cytochrome P450BM3 (P450BMP) were studied versus the concentration of palmitic acid. An increase in hydrostatic pressure causes a high- to low-spin shift and subsequent P450 to P420 transition. Conversion of P450BMP to P420 is associated with important conformational and hydration changes of the protein. Treating the pressure-induced changes in the high-spin content in P450 in terms of the four-state model of spin transitions and substrate binding, we evaluated and compared the barotropic parameters of these transitions for P450MBP, P450cam, and P450 2B4 (2B4). In the current study, the pressure-induced transitions in P450cam were reinvestigated versus the concentration of camphor. The interactions of 2B4 and P450BMP with their substrates (benzphetamine and palmitic acid) were accompanied by larger changes in the partial volume of the proteins (+267 and +248 mL/mol, respectively) than the interactions of P450cam with camphor (+106 mL/mol). For 2B4 and P450BMP, substrate binding apparently requires hydration of regions outside the active site. The reaction volumes of the low- to high-spin transitions of the substrate-free cytochromes (20-23 mL/mol) are consistent with the displacement of one water molecule. The volume changes in the high- to low-spin transition of the substrate-bound P450cam, 2B4, and P450BMP (-90, -49, and -16 mL/mol correspondingly) reveal a linear relationship with DeltaG degrees of the spin transition, suggesting that modulation of the spin state by substrate binding is driven by a common mechanism in all three heme proteins.     

Conformational analysis of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-galactopyranoside by MM and Langevin dynamics simulations

The conformational space of methyl 6-O-[(R)- and (S)-1-carboxyethyl]-alpha-D-galactopyranoside has been investigated. A grid search employing energy minimization at each grid point over the three major degrees of freedom, namely phi, psi and omega, identified low energy regions. The R-isomer shows five low energy conformers within ca. 1 kcal mol(-1) of the global energy minimum. The S-isomer has two conformers within a few tenths of a kcal mol(-1) of the global energy minimum. Langevin dynamics simulations have been have been performed at 300 K for 30 ns of each isomer. The phi dihedral angle has as its major conformer (g-) for the R-isomer whereas it is the (g+) conformer for the S-isomer. For the psi dihedral angle the (t) conformer has the highest population for both isomers. The dihedral angle omega has the (g+) conformer most highly populated, both for the R- and S-isomer. The above five and two conformational states for the R- and S-isomers, respectively, make up 90% in each case of the populated states during the Langevin dynamics (LD) simulations. Rate constants for the omega dihedral angle have been calculated based on a number correlation function. Three bond homo- and heteronuclear, i.e. proton and carbon-13, coupling constants have been calculated from the dynamics trajectories for comparison to experimental values. The heteronuclear coupling constant H2',C6 has been measured for the S-isomer and found to be 3.3 Hz. The J value calculated from the LD simulations, namely 2.6 Hz, is in fair agreement with experiment. A comparison to the X-ray structure of the R-isomer shows that the conformation of the crystalline compound occupies the low energy region most highly populated as a single R-conformer (30%) during the LD simulations.     

Conformational properties of DNA dodecamers containing four tandem repeats of the CNG triplets

We studied DNA dodecamers (CAG)4, (CCG)4, (CGG)4 and (CTG)4by CD spectroscopy and polyacrylamide gel electrophoresis. Each dodecamer adopted several ordered conformers which denatured in a cooperative way. Stability of the conformers depended on the dodecamer concentration, ionic strength, temperature and pH. The dodecamers, having a pyrimidine base in the triplet center, generated foldbacks at low ionic strength whose stem conformations were governed by the GC pairs. At high salt, (CCG)4 isomerized into a peculiar association of two strands. The association was also promoted by high oligonucleotide concentrations. No similar behavior was exhibited by (CTG)4. At low salt, (CGG)4 coexisted in two bimolecular conformers whose populations were strongly dependent on the ionic strength. In addition, (CGG)4 associated into a tetraplex at acidic pH. A tetraplex was even observed at neutral pH if the (CGG)4 concentration was sufficiently high. (CAG)4 was very stable in a monomolecular conformer similar to the known extremely stable foldback of the (GCGAAGC) heptamer. Nevertheless, even this very stable conformer disappeared if (CTG)4 was added to the solution of (CAG)4. Association of the complementary strands was also strongly preferred to the particular strand conformations by the other couple, (CCG)4 and (CGG)4.     

Differential phosphorylation of two size forms of the N-type calcium channel alpha 1 subunit which have different COOH termini

Two size forms of the class B N-type calcium channel alpha 1 subunit were recently identified with CNB1, an antipeptide antibody directed against an intracellular loop of this channel (Westenbroek, R.E., Hell, J.W., Warner, C., Dubel, S.J., Snutch, T.P., and Catterall, W.A. (1992) Neuron 9, 1099-1115). To investigate the biochemical differences between these two size forms, the antibodies CNB3 and CNB4 were raised against peptides with sequences corresponding to the COOH-terminal end of the full-length form. Immunoblot experiments demonstrated that both antibodies specifically recognize the longer form of 250 kDa, indicating that the COOH-terminal regions of the two size forms of the class B N-type channel alpha 1 subunit are different. Phosphorylation experiments with immunopurified calcium channels and different second messenger-activated protein kinases revealed that both the 220- and 250-kDa forms of the class B N-type calcium channel alpha 1 subunit are substrates for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. These three kinases incorporated approximately 1 mol of phosphate/mol of binding sites for omega-conotoxin (omega-CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- and calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated only the long form of the class B N-type calcium channel alpha 1 subunit, with a stoichiometry of 0.5 mol of phosphate/mol of total omega-CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B alpha 1 subunit by CaM kinase II may differentially regulate the function of N-type calcium channels containing different size forms of their alpha 1 subunits in vivo.     

Axial coordination and reduction potentials of the sixteen hemes in high-molecular-mass cytochrome c from Desulfovibrio vulgaris (Hildenborough)

A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.     

Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.     

The origin of differences in the physical properties of the equilibrium forms of cytochrome b5 revealed through high-resolution NMR structures and backbone dynamic analyses

On the basis of a comparison of high-resolution solution structures calculated for both equilibrium forms of rat ferrocytochrome b5, differences in reduction potential and thermodyanmic stability have been characterized in terms of significant structural and dynamic differences between the two forms. The dominant difference between A and B conformations has long been known to be due to a 180 degrees rotation of the heme in the binding pocket about an axis defined by the alpha- and gamma-meso carbons, however, the B form has not been structurally characterized until now. The most significant differences observed between the two forms were the presence of a hydrogen bond between the 7-propionate and the S64 amide in the A form but not the B form and surprisingly a displacement of the heme out of the binding pocket by 0.9 A in the B form relative to the A form. The magnitude of other factors which could contribute to the known difference in reduction potentials in the bovine protein [Walker, F. A., Emrick, D., Rivera, J. E., Hanquet, B. J., and Buttlaire, D. H. (1988) J. Am. Chem. Soc. 110, 6234-6240], such as differences in the orientation of the axial imidazoles and differences in hydrogen bond strength to the imidazoles, have been evaluated. The dominant effector of the reduction potential would appear to be the lack of the hydrogen bond to the S64 amide in the B form which frees up the propionate to charge stabilize the iron in the oxidized state and thus lower the reduction potential of the B form. The structure we report for the A form, based on heteronuclear NMR restraints, involving a total of 1288 restraints strongly resembles both the X-ray crystal structure of the bovine protein and a recently reported structure for the A form of the rat protein based on homonuclear data alone [Banci, L., Bertini, I., Ferroni, F., and Rosato, A. (1997) Eur. J. Biochem. 249, 270-279]. The rmsd for the backbone atoms of the A form is 0.54 A (0.92 A for all non-hydrogens). The rmsd for the backbone of the B form is 0.51 A (0. 90 A for all non-hydrogen atoms). An analysis of backbone dynamics based on a model-free analysis of 15N relaxation data, which incorporated axially symmetric diffusion tensor modeling of the cytochrome, indicates that the protein is more rigid in the reduced state relative to the oxidized state, based on a comparison with order parameters reported for the bovine protein in the oxidized state [Kelly, G. P., Muskett, F. W., and Whitford, D. (1997) Eur. J. Biochem. 245, 349-354].     

Conformational analysis of [Met5]-enkephalin: solvation and ionization considerations

[Met5]-Enkephalin has the sequence Tyr-Gly-Gly-Phe-Met. Only the extended conformation of the peptide has been observed by X-ray crystallography. Nuclear magnetic resonance spectroscopy supports the presence of a turn at Gly 3 and Phe 4 in dimethyl sulfoxide. In this study, the peptide conformational states and thermodynamic properties are understood in terms of ionization state and solvent environment. In the calculation, final conformations obtained from multiple independent Monte Carlo simulated annealing conformational searches are starting points for molecular dynamics simulations. In an aqueous environment given by the use of solvation free energy and the zwitterionic state, dominant structural motifs computed are G-P Type II' bend, G-G Type II' bend, and G-G Type I' bend motifs, in order of increasing free energy. In the calculation of the peptide with neutral N- and C-termini and solvation free energy, the extended conformer dominates (by at least a factor of 2.5), and the conformation of another low free energy conformer superimposes well on the pharmacophoric groups of morphine. Neutralization of charge and solvation induce and stabilize the extended conformation, respectively. A mechanism of inter-conversion between the extended conformer and three bent conformers is supported by phi/psi-scatter plots, and by the conformer relative free energies. An estimate of the entropy change of receptor unbinding is 8.3 cal K-1 mol-1, which gives rise to a -2.5 kcal/mol entropy contribution to the free energy of unbinding at 25 degrees C. The conformational analysis methodology described here should be useful in studies on short peptides and flexible protein surface loops that have important biological implications.     

Flexible ligand docking using conformational ensembles

Molecular docking algorithms suggest possible structures for molecular complexes. They are used to model biological function and to discover potential ligands. A present challenge for docking algorithms is the treatment of molecular flexibility. Here, the rigid body program, DOCK, is modified to allow it to rapidly fit multiple conformations of ligands. Conformations of a given molecule are pre-calculated in the same frame of reference, so that each conformer shares a common rigid fragment with all other conformations. The ligand conformers are then docked together, as an ensemble, into a receptor binding site. This takes advantage of the redundancy present in differing conformers of the same molecule. The algorithm was tested using three organic ligand protein systems and two protein-protein systems. Both the bound and unbound conformations of the receptors were used. The ligand ensemble method found conformations that resembled those determined in X-ray crystal structures (RMS values typically less than 1.5 A). To test the method's usefulness for inhibitor discovery, multi-compound and multi-conformer databases were screened for compounds known to bind to dihydrofolate reductase and compounds known to bind to thymidylate synthase. In both cases, known inhibitors and substrates were identified in conformations resembling those observed experimentally. The ligand ensemble method was 100-fold faster than docking a single conformation at a time and was able to screen a database of over 34 million conformations from 117,000 molecules in one to four CPU days on a workstation.     

2H( gamma,p)n cross section between 20 and 440 MeV

Rate control in acetylcholinesterase (AChE) involves a single anionic site whose anionic center controls rate-related biochemical and conformational changes in the E (free enzyme) and EA (acylated enzyme) conformers. Change in conformer structure and biochemistry affect binding, acylation, and hydrolysis. It is significant that the anionic-esteratic intersite distance is not altered during conformer change as E is converted to EA. In this enzyme system, cationic acetylcholine and anionic AChE are true structural, functional, and biochemical counterparts. The anionic center in the E conformer lies at the bottom of a sterically restricted, hydrophobic cleft < 8 A wide at the top and > 3 A wide at the bottom, while the anionic center in the EA conformer is relatively open. It is characterized by a decrease in the relative binding of hydrophobic cations and by an ability to bind large organic cations. Binding of acetylcholine, H+, or organic cations at the anionic site controls k2(acylation) in the E conformer and k3(hydrolysis) in the EA conformer. Acetylcholine binding forms the ES complex in which the cation maximizes k2. In the EAS complex, the cation reduces k3 and provides allosteric control. Anionic site structure and biochemistry and the effect of pH on k2 and k3 differentiates AChE from butyrylcholinesterase. This comprehensive study of kinetic and thermodynamic processes in AChE was made possible by the synthesis and/or use of families of over 30 cationic and acylation probes of known stereochemistry. They act as rulers of the E and EA conformers of AChE and provide comparative data on kinetic-based and thermodynamic-based constants. Cationic inhibitors affect decarbamylation rates in AChE and provide an additional set of comparative data related to the mechanism of substrate hydrolysis by AChE. Acridine araphanes are unique neural receptor and cholinergic enzyme probes. Their parallel plane and coplanar conformations are related to bridge length. Two parallel plane acridine araphanes are pure uncompetitive inhibitors of AChE. Scatchard plots of the binding of methylacridinium and 9-aminoacridine with the E conformer and 9-aminoacridine with the EA conformer indicate binding at a single anionic site. No ternary complex (EII or EAII) from two-site binding was detected. In AChE, nonspecific, low-level binding at surface ionic and hydrophobic areas is ubiquitous. Binding affinity differences greater than two orders of magnitude distinguish binding at the anionic site from low level binding at surface moieties. Surface binding provides environmental and stability changes in the enzyme but does not modify the fundamental biochemistry of the E and EA conformers.     

Ab initio calculations on (Z)-5-decenyl acetate, a component of the pheromone complex of Agrotis segetum (Lepidoptera: Noctuidae) and electrophysiological studies with chain elongated analogues

Conformational analyses of (Z)-5-decenylacetate, a sex pheromone component of the turnip moth, Agrotis segetum, and double unsaturated pheromone analogues 4 and 5 have been performed by ab initio calculations using Gaussian 92. Two minima were found for a cisoid and a transoid conformer, differing for 0.03 kcal/mol only. Conformational energies of diene analogues (5Z,7E)-5,7-decadienyl acetate (4) and (3E,5Z)-3,5,-decadienyl acetate (5) were determined for conformers required to mimic spatial relationships of the cisoid conformation of the natural pheromone 2. Finally, single sensillum recording studies were carried out with chain elongated C11- to C16-pheromone analogues 6.     

Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy

Time-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site.     

Conformation of xanthene dyes in the sulfhydryl 1 binding site of myosin. 2

The fluorescent dyes 5'-(iodoacetamido)tetramethylrhodamine (5'IATR) and 5'-(iodoacetamido)-fluorescein (5'IAF) bind covalently to the reactive sulfhydryl (SH1) of myosin subfragment 1 (S1), the 5'IATR as a dimer and the 5'IAF as a monomer. The conformation of the dimer and the dye-protein complex was investigated by comparison of several spectroscopic signals of the molecules before and after their association into a complex and interpretation of any changes using a coupled dipole oscillator model adapted for this problem [Burghardt & Ajtai (1995) Biophys. Chem. (submitted for publication)]. Absorption and fluorescence spectroscopies were performed on 5'IAF, 5'IATR, and rhodamine 6G (R6G) and rhodamine B (RB) as models of dimer conformation. Absorption, fluorescence, and circular dichroism (CD) spectroscopies were performed on 5'IATR-modified S1 (5'R-S1) and 5'IAF-modified S1 (5'F-S1). Combined spectroscopic and 2-D NMR data from rhodamines in solution determined the conformations of the dimers. Xanthene rings from dimers of identical dyes (homodimers) stacked in two structures having very different spectroscopic signatures. Xanthene rings from the heterodimer of R6G and RB stacked in one conformation. The two homodimer conformations of 5'IATR are equally likely to form in solution. The other rhodamine homodimers have one dominant, but not exclusive, structure. Both conformations of the 5'IATR dimer were coupled to a tryptophan as a model of the dye-protein interaction at SH1. The calculated CD from one dimer conformer (dimer A) coupled to tryptophan is negative for the lowest energy CD absorption band. The other dimer (dimer B) gives positive CD on the two lowest energy CD absorption bands. Both dimer structures of 5'IATR contributed to the early time-dependent CD signal from 5'IATR binding to SH1, but at equilibrium the CD signal indicated only dimer B, suggesting that the SH1 binding pocket converts dimer A into dimer B. The time-dependent CD signal from 5'IAF changes amplitude but not shape during the reaction with SH1. The model calculation accounting for the spectroscopic signals of 5'R-S1 and 5'F-S1 indicates several likely conformations of the 5'IATR dimer-tryptophan and 5'IAF-tryptophan complexes embedded in S1. These structures fit to the alpha-carbon structure of the SH1 binding pocket when the 5'IATR dimer and 5'IAF interact closely with Trp510 [Rayment et al. (1993) Science 261, 50-58].(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.     

Serum soluble IL-6 receptor concentrations correlate with stages of multiple myeloma defined by serum beta 2-microglobulin and C-reactive protein

Serum soluble interleukin-6 receptor (sIL-6R) concentrations were measured in 52 patients with multiple myeloma (MM) and 24 normal controls, using a commercially available immunoenzymatic assay kit. Patients were staged according to the Bataille et al. myeloma staging system based on the levels of patients' serum beta 2-microglobulin and C-reactive protein. Twenty-one patients were at stage A of disease, 19 at stage B and 12 at stage C at the time of serum collection for sIL-6R determination. Serum sIL-6R concentrations ranged from 15 to 176 ng/ml with a mean of 64.8 +/- 35.9 ng/ml and a median of 58 ng/ml in the entire group of patients studied. These values were significantly higher than those of 34.4 +/- 13.4 ng/ml found in the controls (P < or = 0.001). Patients of stage C had higher sIL-6R levels (94.8 + 41.2 ng/ml) than patients of stage B (67.7 +/- 31.0 ng/ml) (P < 0.01), and markedly higher than patients of stage A (45.0 +/- 23.1 ng/ml) (P < 0.001). Serum levels of sIL-6R in patients with stage A disease did not differ statistically from those of the controls. A linear positive correlation was observed between serum levels of the receptor and the stage of MM (r = 0.539, P < 0.001). These data strongly suggest that serum sIL-6R concentrations correlate with the stages of MM and may be used as an indicator of the activity of the disease.     

A slipped replication intermediate model is stabilized by the syn orientation of N-2-aminofluorene- and N-2-(acetyl)aminofluorene-modified guanine at a mutational hotspot

The Escherichia coli NarI restriction enzyme recognition site 5'G1G2C3G4C5C63' is a mutational hotspot for -2 deletions in E. coli plasmid pBR322, resulting in the sequence 5'GGCC3' when G4 is modified by the aromatic amine N-2-(acetyl)aminofluorene (AAF) [Burnouf, D., Koehl, P., and Fuchs, R. P. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 86, 4147-4151] even though each G shows similar reactivity [Fuchs, R. P. P. (1984) J. Mol. Biol. 177, 173-180]. Modification at G4 by the related aromatic amine 2-aminofluorene (AF), which lacks the acetyl group of AAF, can also cause -2 deletions, but at a lower frequency [Bichara, M., and Fuchs, R. P. P. (1985) J. Mol. Biol. 183, 341-351]. A specific mechanism has been proposed to explain the double-base frameshifts in the NarI sequence in which the GC deletion results from a slipped mutagenic intermediate formed during replication [Schaaper, B. M., Koffel-Schwartz, N., and Fuchs, R. P. P. (1990) Carcinogenesis 11, 1087-1095]. We address the following key questions in this study. Why does AAF modification dramatically increase the mutagenicity at the NarI G4 position, and why does AAF enhance the mutagenicity more than AF? We studied two intermediates which model replication at one arm of a fork, using a fragment of DNA modified by AF or AAF at G4 in the NarI sequence: Intermediate I can be converted into intermediate II by misalignment. Elongation of intermediate I leads to error-free translesion synthesis, while elongation of intermediate II leads to a -2 frameshift mutation. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to investigate the conformations of the AF and AAF adducts at G4 in these two intermediates. We find that the slipped mutagenic intermediate is quite stable relative to its normally extended counterpart in the presence of AF and AAF in an abnormal syn orientation of the damaged base. An enhanced probability of elongation from a stable slipped structure rather than a properly aligned one would favor increased -2 frameshift mutations. Furthermore, AAF-modified DNA has a greater tendency to adopt the syn orientation than AF because of its greater bulk, which could explain its greater propensity to cause -2 deletions in the NarI sequence.