One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

L p boundedness of Marcinkiewicz integrals with H 1 kernels

We establish the L^p boundedness of Marcinkiewicz integral operators with kernels in theHardy space on a compact submanifold of finite type. Previously such boundedness properties were obtained when the submanifold is a sphere. Our result shows that what matters is not the symmetry, but a certain type of curvature property of the submanifold.     

MAOS ls for the general synthesis and lead optimization of 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines

General, high-yielding MAOS protocols for the expedient synthesis of functionalized 3,6-disubstituted-[1,2,4]triazolo[4,3-b]pyridazines are described amenable to an iterative analog library synthesis strategy for the lead optimization of an M1 antagonist screening hit. Optimized compounds proved to be highly selective M1 antagonists.     

A study on the interactions of Aurein 2.5 with bacterial membranes

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH₂) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25 mN m⁻¹) and molecular areas consistent with the adoption of α-helical structure orientated either perpendicular (1.72 nm² molecule⁻¹) or parallel (3.6 nm² molecule⁻¹) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30 μM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9 mN m⁻¹ and 5 mN m⁻¹, respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed ΔG_Mix > 0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated α-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5 mN m⁻¹) and DOPE monolayers (6 mN m⁻¹) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125 μM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.     

Volume-based thermoelasticity: consequences of the (near) proportionality of isothermal compressibility to formula-unit volume

Groups of structurally related materials, including the alkali halides, exhibit a proportionality of isothermal compressibility to formula-unit volume. The relationship has recently been explored by Glasser and by Recio et al. In this paper, we present the consequences of such proportionality on the relationships of Born-Lande? and Born-Mayer parameters to the formula-unit volume. These relationships have then been tested separately on (i) alkali (excluding cesium) halides and (ii) cesium halides. We conclude that the equations fit the NaCl-type materials satisfactorily, but less well for the CsCl-type materials, and that the Born-Mayer equation is more applicable. These results confirm the conclusion that volume is intimately linked to thermodynamic quantities, as already demonstrated by our development of volume-based thermodynamics (VBT).     

Rapid access to structured triacylglycerols acylated with n-3 polyunsaturated fatty acids for nutritional applications

In order to better understand the metabolic fate of n-3 polyunsaturated fatty acids (PUFAs), an efficient access to symmetrical and unsymmetrical triacylglycerols (TGs), esterified with PUFAs, with known high purity, is required. In this context, we optimized the esterification of a mixture of glycerols protected as dioxane and dioxolane with PUFAs. The kinetics of this reaction depends on various factors, such as the fatty acid chain length and the stereochemistry of the dioxane. Then, one-pot acetal hydrolysis and esterification of hydroxyl groups led to the desired structured TGs without either double bond isomerization or acyl migration (except when symmetrical TGs are acylated with long-chain saturated fatty acids in external positions). PUFAs location on the glycerol backbone was assayed by NMR, HPLC and pancreatic lipase hydrolysis.     

Lateralization produced by envelope-based interaural temporal disparities of high-frequency, raised-sine stimuli: empirical data and modeling

An acoustic pointing task was used to measure extents of laterality produced by ongoing interaural temporal disparities (ITDs) conveyed by the envelopes of 4-kHz-centered raised-sine stimuli while varying, parametrically, their peakedness, depth of modulation, and frequency of modulation. One purpose of the study was to determine whether such manipulations would produce changes in laterality logically consistent with those found for ITD-discrimination thresholds reported by Bernstein and Trahiotis [J. Acoust. Soc. Am. 125, 3234-3242 (2009)]. The data obtained revealed that they did in that (1) increasing depth of modulation, peakedness, or frequency of modulation between 32 and 128 Hz produced smaller threshold ITDs and greater laterality and (2) increasing frequency of modulation to 256 Hz produced modest increases in threshold ITDs and modest decreases in laterality. The extents of laterality measured were successfully accounted for via an augmentation of the cross-correlation-based "position-variable" modeling approach developed by Stern and Shear [J. Acoust. Soc. Am. 100, 2278-2288 (1996)] to account for ITD-based extents of laterality obtained at low spectral frequencies.     

Glycerol and glycerol carbonate as ultraviscous solvents for mixture analysis by NMR

NMR of weakly polar analytes in an apolar ultraviscous solvent has recently been proposed for mixture analysis as a pertinent alternative to the DOSY experiment. The present article reports the first use of glycerol and glycerol carbonate as polar solvents for the NMR analysis of a model mixture of dipeptides. This work demonstrates the high potentiality of these solvents for the analysis of mixtures made of polar and potentially bioactive compounds. Medium-sized molecules slowly reorient in glycerol and glycerol carbonate under particular temperature conditions, so that solute resonances may show spin diffusion in NOESY spectra, thus opening the way to mixture analysis. Glycerol and glycerol carbonate have turned out to be ultraviscous solvents of choice for the individualization of four structurally close mixed dipeptides: Leu-Val, Leu-Tyr, Gly-Tyr and Ala-Tyr by means of 1D and 2D NOESY experiments. Selective sample excitation and signal detection were implemented to eliminate the intense proton signals of the non-deuterated solvents. Moreover, the recording of a multiplet selective 2D NOESY-TOCSY has shown that the analytical power of NMR in highly viscous solvents is not limited to the extraction of mixture component 1D subspectra but may also yield some supplementary information about atom connectivity within components.