Solid-state 2H NMR structure of retinal in metarhodopsin I

The structural and photochemical changes in rhodopsin due to absorption of light are crucial for understanding the process of visual signaling. We investigated the structure of trans-retinal in the metarhodopsin I photointermediate (MI), where the retinylidene cofactor functions as an antagonist. Rhodopsin was regenerated using retinal that was (2)H-labeled at the C5, C9, or C13 methyl groups and was reconstituted with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Membranes were aligned by isopotential centrifugation, and rhodopsin in the supported bilayers was then bleached and cryotrapped in the MI state. Solid-state (2)H NMR spectra of oriented rhodopsin in the low-temperature lipid gel state were analyzed in terms of a static uniaxial distribution (Nevzorov, A. A.; Moltke, S.; Heyn, M. P.; Brown, M. F. J. Am. Chem. Soc. 1999, 121, 7636-7643). The line shape analysis allowed us to obtain the methyl bond orientations relative to the membrane normal in the presence of substantial alignment disorder (mosaic spread). Relative orientations of the methyl groups were used to calculate effective torsional angles between the three different planes that represent the polyene chain and the beta-ionone ring of retinal. Assuming a three-plane model, a less distorted structure was found for retinal in MI compared to the dark state. Our results are pertinent to how photonic energy is channeled within the protein to allow the strained retinal conformation to relax, thereby forming the activated state of the receptor.     

Drug monitoring and toxicology: a procedure for the monitoring of oxcarbazepine metabolite by HPLC-UV

This article describes a rapid high-performance liquid chromatographic (HPLC) method for the measurement of the primary metabolite of oxcarbazepine. Following a simple precipitation step, 10,11,-dihydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide is quantitated (5-60 microg/mL) by analysis on an HPLC-UV system. The instrument time is less than 5 min per injection, an improvement over most published methods. The assay's limit of quantitation, linearity, imprecision, and accuracy adequately cover the therapeutic range for appropriate patient monitoring. In comparison to other published methods, this procedure would be of interest to clinical laboratories because it employs a precipitation step for sample preparation, instead of conventional yet time-consuming solid-phase extraction.     

Unraveling the aflatoxin-FAPY conundrum: structural basis for differential replicative processing of isomeric forms of the formamidopyrimidine-type DNA adduct of aflatoxin B1

Aflatoxin B1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [Smela, M. E.; Hamm, M. L.; Henderson, P. T.; Harris, C. M.; Harris, T. M.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6655-6660]. NMR studies carried out on the AFB-FAPY bases and deoxynucleoside 3',5'-dibutyrates now establish that the separable FAPY bases and nucleosides are diastereomeric N5 formyl derivatives involving axial asymmetry around the congested pyrimidine C5-N5 bond. Anomerization of the protected beta-deoxyriboside was not observed, but in the absence of acyl protection, both anomerization and furanosyl --> pyranosyl ring expansion occurred. In oligodeoxynucleotides, two equilibrating FAPY species, separable by HPLC, are assigned as anomers. The form normally present in duplex DNA is the mutagenic species. It has previously been assigned as the beta anomer by NMR (Mao, H.; Deng, Z. W.; Wang, F.; Harris, T. M.; Stone, M. P. Biochemistry 1998, 37, 4374-4387). In single-stranded environments the dominant species is the beta anomer; it is a block to replication.     

Potentiometric titration of metal ions in ethanol

The potentiometric titrations of Zn2+, Cu2+ and 12 Ln3+ metal ions were obtained in ethanol to determine the titration constants (defined as the at which the [-OEt]/[Mx+]t ratios are 0.5, 1.5, and 2.5) and in two cases (La3+ and Zn2+) a complete speciation diagram. Several simple monobasic acids and aminium ions were also titrated to test the validity of experimental titration measurements and to establish new constants in this medium that will be useful for the preparation of buffers and standard solutions. The dependence of the titration constants on the concentration and type of metal ion and specific counterion effects is discussed. In selected cases, the titration profiles were analyzed using a commercially available fitting program to obtain information about the species present in solution, including La3+ for which a dimer model is proposed. The fitting provides the microscopic values for deprotonation of one to four metal-bound ethanol molecules. Kinetics for the La3+-catalyzed ethanolysis of paraoxon as a function of are presented and analyzed in terms of La3+ speciation as determined by the analysis of potentiometric titration curves. The stability constants for the formation of Zn2+ and Cu2+ complexes with 1,5,9-triazacyclododecane as determined by potentiometric titration are presented.     

Nonequilibrium membrane fluctuations driven by active proteins

We extend a model for nonthermal membrane undulations driven by active (adenosine triphosphate-dependent or light-harvesting) membrane proteins [N. Gov, Phys. Rev. Lett. 93, 268104 (2004)]. The present model accounts for the fact that proteins can diffuse laterally across the membrane surface and that individual proteins are expected to exert forces preferentially in one normal direction over the other (due to their orientation within the bilayer). The addition of these effects alters the scaling of fluctuation amplitudes with system size. Additionally, theoretical arguments and dynamic simulations both suggest that, in certain regimes, the probability distribution of fluctuation amplitudes is expected to be non-Gaussian (in contrast to thermal systems).     

Kinetics and mechanism of ketone enolization mediated by magnesium bis(hexamethyldisilazide)

Magnesium bis(hexamethyldisilazide), Mg(HMDS)(2), reacts with substoichiometric amounts of propiophenone in toluene solution at ambient temperature to form a 74:26 mixture of the enolates (E)- and (Z)-[(HMDS)(2)Mg(2)(mu-HMDS){mu-OC(Ph)=CHCH(3)}], (E)-1 and (Z)-1, which contain a pair of three-coordinate metal centers bridged by an amide and an enolate group. The compositions of (E)-1 and (Z)-1 were confirmed by solution NMR studies and also by crystallographic characterization in the solid state. Rate studies using UV-vis spectroscopy reveal the rapid and complete formation of a reaction intermediate, 2, between the ketone and magnesium, which undergoes first-order decay with rate constants independent of the concentration of excess Mg(HMDS)(2) (DeltaH++ = 17.2 +/- 0.8 kcal/mol, DeltaS++ = -11 +/- 3 cal/mol.K). The intermediate 2 has been characterized by low-temperature (1)H NMR, diffusion-ordered NMR, and IR spectroscopy and investigated by computational studies, all of which are consistent with the formulation of 2 as a three-coordinate monomer, (HMDS)(2)Mg{eta(1)-O=C(Ph)CH(2)CH(3)}. Further support for this structure is provided by the synthesis and structural characterization of two model ketone complexes, (HMDS)(2)Mg(eta(1)-O=C(t)Bu(2)) (3) and (HMDS)(2)Mg{eta(1)-O=C((t)Bu)Ph} (4). A large primary deuterium isotope effect (k(H)/k(D) = 18.9 at 295 K) indicates that proton transfer is the rate-limiting step of the reaction. The isotope effect displays a strong temperature dependence, indicative of tunneling. In combination, these data support the mechanism of enolization proceeding through the single intermediate 2 via intramolecular proton transfer from the alpha carbon of the bound ketone to the nitrogen of a bound hexamethyldisilazide.     

Combination of a dinuclear Zn2+ complex and a medium effect exerts a 10(12)-fold rate enhancement of cleavage of an RNA and DNA model system

The catalytic ability of a dinuclear Zn2+ complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)propane (3) in promoting the cleavage of an RNA model, 2-hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1), and a DNA model, methyl p-nitrophenyl phosphate (MNPP, 4), was studied in methanol solution in the presence of added CH3O- at 25 degrees C. The di-Zn2+ complex (Zn2 :3), in the presence of 1 equiv of added methoxide, exhibits a second-order rate constant of (2.75 +/- 0.10) x 10(5) M(-1) s(-1) for the reaction with 1 at s(s)pH 9.5, this being 10(8)-fold larger than the k2 value for the CH3O- promoted reaction (kOCH3 = (2.56 +/- 0.16) x 10(-3) M(-1) s(-1)). The complex is also active toward the DNA model 4, exhibiting Michaelis-Menten kinetics with a KM and kmax of 0.37 +/- 0.07 mM and (4.1 +/- 0.3) x 10(-2) s(-1), respectively. Relative to the background reactions at s(s)pH 9.5, Zn2 :3 accelerates cleavage of each phosphate diester by a remarkable factor of 1012-fold. A kinetic scheme common to both substrates is discussed. The study shows that a simple model system comprising a dinuclear Zn2+ complex and a medium effect of the alcohol solvent achieves a catalytic reactivity that approaches enzymatic rates and is well beyond anything seen to date in water for the cleavage of these phosphate diesters.     

NMR observations of 13C-enriched coenzyme B12 bound to the ribonucleotide reductase from Lactobacillus leichmannii

The 13C NMR resonance and one-bond 1H-13C coupling constants of coenzyme B12 enriched in 13C in the cobalt-bound carbon have been observed in the complex of the coenzyme with the B12-dependent ribonucleotide reductase from Lactobacillus leichmannii. Neither the 13C NMR chemical shift nor the 1H-13C coupling constants are significantly altered by binding of the coenzyme to the enzyme. The results suggest that ground-state Co-C bond distortion is not utilized by this enzyme to activate coenzyme B12 for C-Co bond homolysis.     

Sample-specific diamagnetic and paramagnetic passive shimming

When homogenizing the static magnetic field over extended in vivo volumes, significant residual inhomogeneity can remain after spherical harmonic shim optimization. This is due to the low spatial orders of shims available on in vivo MR systems and the presence of higher-order inhomogeneity in the vicinity of anatomic air cavities. Mediation of this problem through the development of higher-order spherical harmonic shims is severely impeded by bore space limitations. Sample-specific passive shims are not limited to low-order spatial compensation and offer an alternative means to increased homogenization. Here, we present a novel construction protocol for sample-specific passive shims comprised of both diamagnetic (bismuth) and paramagnetic (zirconium) materials. A prototype shim is constructed and shown to significantly homogenize the mouse brain at 9.4 T. Further homogenization capabilities are simulated through alteration of the shim construction.