Negative nuclear Overhauser effects as probes of macromolecular structure

Nuclear Overhauser effects (NOE) and circular dichroism (CD) techniques have been used to probe @-turn conformations in acyclic and cyclic peptides containingPro-Xsequences. The model peptides studied are of the type Piv-Pro-X-NHMe (X = Aib, D-Ala, Gly, Val, and Leu) and Boc-Cys-Pro-X-C s NHMe (X = Aib, L-Ala, D-Ala, Gly, and Leu). In the acyclic series, observation of NOES between Pro C"H and X-NH, together with solvent and temperature dependence of NH chemical shifts, establishes a 4 - 1 hydrogen bond stabilized type I1 @-turn in the Gly, D-Ala, and Aib peptides, in CDC13 and (CD3)2S0. A positive n-r* CD band at -225-230 nm appears to be characteristic of this structure. For the acyclic Pro-Leu peptide the observation of NOE's for both Pro and Leu C"H resonances on saturation of Leu NH is compatible with a type V bend or consecutive y-turn conformation. In the cyclic disulfide series the Pro-Aib and Pro-D-Ala peptides favor type I1 @-turns, whereas all other peptides adopt type I (111) conformations. All the cyclic disulfides exhibit an intense negative CD band at -228-230 nm. The results suggest thatgeneralcorrelations between CD spectral type and specific 0-turn conformations may not be obtained. Evidence for solvent-dependent structural changes in the Pro-Aib sequence in both cyclic and acyclic peptides is presented.

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motio

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

Multiple conformational states in crystals and in solution in alpha gamma hybrid peptides. Fragility of the C-12 helix in short sequences

The conformational properties of foldamers generated from alpha gamma hybrid peptide sequences have been probed in the model sequence Boc-Aib-Gpn-Aib-Gpn-NHMe. The choice of alpha-aminoisobutyryl (Aib) and gabapentin (Gpn) residues greatly restricts sterically accessible coil formational space. This model sequence was anticipated to be a short segment of the alpha gamma C-12 helix, stabilized by three successive 4 -> 1 hydrogen bonds, corresponding to a backbone-expanded analogue of the alpha polypeptide 3(10)-helix. Unexpectedly, three distinct crystalline polymorphs were characterized in the solid state by X-ray diffraction. In one form, two successive C-12 hydrogen bonds were obtained at the N-terminus, while a novel C-17 hydrogen-bonded gamma alpha gamma turn was observed at the C-terminus. In the other two polymorphs, isolated C-9 and C-7 hydrogen-bonded turns were observed at Gpn (2) and Gpn (4). Isolated C-12 and C-9 turns were also crystallographically established in the peptides Boc-Aib-Gpn-Aib-OMe and Boc-Gpn-Aib-NHMe, respectively. Selective line broadening of NH resonances and the observation of medium range NH(i)<-> NH(i+2) NOEs established the presence of conformational heterogeneity for the tetrapeptide in CDCl3 solution. The NMR results are consistent with the limited population of the continuous C-12 helix conformation. Lengthening of the (alpha gamma)(n) sequences in the nonapeptides Boc-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Xxx (Xxx = Aib, Leu) resulted in the observation of all of the sequential NOEs characteristic of an alpha gamma C-12 helix. These results establish that conformational fragility is manifested in short hybrid alpha gamma sequences despite the choice of conformationally constrained residues, while stable helices are formed on chain extension.

Analysis of dynamics and mechanism of ligand binding to Artocarpus integrifolia agglutinin. A 13C and 19F NMR study

Binding of 13C-labeled N-acetylgalactosamine (13C-GalNAc) and N-trifluoroacetylgalactosamine (19F-GalNAc) to Artocarpus integrifolia agglutinin has been studied using 13C and 19F nuclear magnetic resonance spectroscopy, respectively. Binding of these saccharides resulted in broadening of the resonances, and no change in chemical shift was observed, suggesting that the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc experience a magnetically equivalent environment in the lectin combining site. The alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc were found to be in slow exchange between free and protein bound states. Binding of 13C-GalNAc was studied as a function of temperature. From the temperature dependence of the line broadening, the thermodynamic and kinetic parameters were evaluated. The association rate constants obtained for the alpha-anomers of 13C-GalNAc and 19F-GalNAc (k+1 = 1.01 x 10(5) M-1.s-1 and 0.698 x 10(5) M-1.s-1, respectively) are in close agreement with those obtained for the corresponding beta-anomers (k+1 = 0.95 x 10(5) M-1.s-1 and 0.65 x 10(5) M-1.s-1, respectively), suggesting that the two anomers bind to the lectin by a similar mechanism. In addition these values are several orders of magnitude slower than those obtained for diffusion controlled processes. The dissociation rate constants obtained are 49.9, 56.9, 42, and 43 s-1, respectively, for the alpha- and beta-anomers of 13C-GalNAc and 19F-GalNAc. A two-step mechanism has been proposed for the interaction of 13C-GalNAc and 19F-GalNAc with A. integrifolia lectin in view of the slow association rates and high activation entropies. The thermodynamic parameters obtained for the association and dissociation reactions suggest that the binding process is entropically favored and that there is a small enthalpic contribution.

Stabilizing hadron resonance gas models

We examine the stability of hadron resonance gas models by extending them to include undiscovered resonances through the Hagedorn formula. We find that the influence of unknown resonances on thermodynamics is large but bounded. We model the decays of resonances and investigate the ratios of particle yields in heavy-ion collisions. We find that observables such as hydrodynamics and hadron yield ratios change little upon extending the model. As a result, heavy-ion collisions at the RHIC and LHC are insensitive to a possible exponential rise in the hadronic density of states, thus increasing the stability of the predictions of hadron resonance gas models in this context. Hadron resonance gases are internally consistent up to a temperature higher than the crossover temperature in QCD, but by examining quark number susceptibilities we find that their region of applicability ends below the QCD crossover.

Type I seesaw mechanism for quasi-degenerate neutrinos

We discuss symmetries and scenarios leading to quasi-degenerate neutrinos in type I seesaw models. The existence of degeneracy in the present approach is not linked to any specific structure for the Dirac neutrino Yukawa coupling matrix y(D) and holds in general. Basic input is the application of the minimal flavour violation principle to the leptonic sector. Generalizing this principle, we assume that the structure of the right-handed neutrino mass matrix is determined by y(D) and the charged lepton Yukawa coupling matrix y(l) in an effective theory invariant under specific groups G(F) contained in the full symmetry group of the kinetic energy terms. G(F) invariance also leads to specific structure for the departure from degeneracy. The neutrino mass matrix (with degenerate mass m(0)) resulting after seesaw mechanism has a simple form Mv approximate to m(0)(I - py(l)y(l)(T)) in one particular scenario based on supersymmetry. This form is shown tolead to correct description of neutrino masses and mixing angles. The thermal leptogenesis after inclusion of flavour effects can account for the observed baryon asymmetry of the universe within the present scenario. Rates for lepton flavour violating processes can occur at observable levels in the supersymmetric version of the scenario. (c) 2010 Elsevier B.V. All rights reserved.

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (N alpha-Fmoc-Ser-[Ac-4,-beta-D-Gal-(1,3)-Ac(2)alpha-D-GalN(3)]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D H-1 NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, C alpha H chemical shift perturbations, (3)J(NH:C alpha H) couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.

Plasticity in the primary binding site of galactose/N-acetylgalactosamine-specific lectins - Implication of the C-H center dot center dot center dot O hydrogen bond at the specificity-determining C-4 locus of the saccharide in 4-methoxygalactose recognition by jacalin and winged bean (basic) agglutinin I

It is currently believed that an unsubstituted axial hydroxyl at the specificity-determining C-4 locus of galactose is indispensable for recognition by galactose/N-acetylgalactosamine-specific lectins. Titration calorimetry demonstrates that 4-methoxygalactose retains binding allegiance to the Moraceae lectin jacalin and the Leguminosae lectin, winged bean (basic) agglutinin (WBA I). The binding reactions were driven by dominant favorable enthalpic contributions and exhibited significant enthalpy-entropy compensation. Proton NMR titration of C-methoxygalactose with jacalin and WBA I resulted in broadening of the sugar resonances without any change in chemical shift. The alpha-and beta-anomers of 4-methoxygalactose were found to be in slow exchange with free and lectin-bound states. Both the anomers experience magnetically equivalent environments at the respective binding sites. The binding constants derived from the dependence of NMR line widths on 4-methoxygalactose concentration agreed well with those obtained from titration calorimetry. The results unequivocally demonstrate that the loci corresponding to the axially oriented C-4 hydroxyl group of galactose within the primary binding site of these lectins exhibit plasticity. These analyses suggest, for the first time, the existence of C-H ... O-type hydrogen-bond(s) in protein-carbohydrate interactions in general and between the C-4 locus of galactose derivative and the lectins jacalin and WBA I in particular.

Superharmonic vibrations of order 3 in stretched strings

Superharmonic vibrations of order 3 in stretched strings driven by a single-mode planar simple-harmonic force are investigated. It is shown that planar as well as nonplanar superharmonic resonances can occur. After giving a few analytical results, the problem is thoroughly investigated numerically. The stability analysis shows that the region of stable nonplanar oscillations is much smaller than that of stable planar oscillation. It is observed in the case of nonplanar oscillations that the amplitude of the superharmonic in the plane normal to the plane of excitation is larger than that in the plane of excitation.

On measurement of top polarization as a probe of t(t)over-bar production mechanisms at the LHC

In this note we demonstrate the use of top polarization in the study of t (t) over bar resonances at the LHC, in the possible case where the dynamics implies a non-zero top polarization. As a probe of top polarization we construct an asymmetry in the decay-lepton azimuthal angle distribution (corresponding to the sign of cos phi(l)) in the laboratory. The asymmetry is non-vanishing even for a symmetric collider like the LHC, where a positive z axis is not uniquely defined. The angular distribution of the leptons has the advantage of being a faithful top-spin analyzer, unaffected by possible anomalous tbW couplings, to linear order. We study, for purposes of demonstration, the case of a Z' as might exist in the little Higgs models. We identify kinematic cuts which ensure that our asymmetry reflects the polarization in sign and magnitude. We investigate possibilities at the LHC with two energy options: root s = 14TeV and root s = 7TeV, as well as at the Tevatron. At the LHC the model predicts net top quark polarization of the order of a few per cent for M-Z' similar or equal to 1200GeV, being as high as 10% for a smaller mass of the Z' of 700GeV and for the largest allowed coupling in the model, the values being higher for the 7TeV option. These polarizations translate to a deviation from the standard-model value of azimuthal asymmetry of up to about 4% (7%) for 14 (7) TeV LHC, whereas for the Tevatron, values as high as 12% are attained. For the 14TeV LHC with an integrated luminosity of 10 fb(-1), these numbers translate into a 3 sigma sensitivity over a large part of the range 500 less than or similar to M-Z' less than or similar to 1500GeV.

13 C nuclear magnetic resonance studies of the binding of alkali and alkaline earth metal salts to amides

13 C resonances of carbonyl and methyl groups in amides are shifted down-field on interaction with alkali and alkaline earth metal salts. The magnitude of the shift depends on the ionic potential of the cation. Ions like Li+ bind to the amide carbonyl group both in neat amide solutions as well as in concentrated salt solutions in water.

Proton Magnetic-Resonance Study of Cycloheximide-Ribosome Interactions

Cycloheximide-ribosome interactions from sensitive and resistant organisms were studied by proton magnetic resonance spectroscopic techniques. The two methyl resonances of cycloheximide upon interaction with ribosomes from Saccharomyces cerevisiae showed preferential broadening. Comparison of cycloheximide line broadening as effected by ribosomes from S. cerevisiae (sensitive) and Microsporum canis (resistant) revealed that less cycloheximide is bound to the M. canis ribosomes. From the decrease in line broadening observed with increasing temperature it may be concluded that cycloheximide-ribosome interaction is a fast exchange reaction. Tetracycline did not compete with cycloheximide for binding site(s) on the ribosomes of S. cerevisiae.

Amino acid selective unlabeling for sequence specific resonance assignments in proteins

Sequence specific resonance assignment constitutes an important step towards high-resolution structure determination of proteins by NMR and is aided by selective identification and assignment of amino acid types. The traditional approach to selective labeling yields only the chemical shifts of the particular amino acid being selected and does not help in establishing a link between adjacent residues along the polypeptide chain, which is important for sequential assignments. An alternative approach is the method of amino acid selective `unlabeling' or reverse labeling, which involves selective unlabeling of specific amino acid types against a uniformly C-13/N-15 labeled background. Based on this method, we present a novel approach for sequential assignments in proteins. The method involves a new NMR experiment named, {(CO)-C-12 (i) -N-15 (i+1)}-filtered HSQC, which aids in linking the H-1(N)/N-15 resonances of the selectively unlabeled residue, i, and its C-terminal neighbor, i + 1, in HN-detected double and triple resonance spectra. This leads to the assignment of a tri-peptide segment from the knowledge of the amino acid types of residues: i - 1, i and i + 1, thereby speeding up the sequential assignment process. The method has the advantage of being relatively inexpensive, applicable to H-2 labeled protein and can be coupled with cell-free synthesis and/or automated assignment approaches. A detailed survey involving unlabeling of different amino acid types individually or in pairs reveals that the proposed approach is also robust to misincorporation of N-14 at undesired sites. Taken together, this study represents the first application of selective unlabeling for sequence specific resonance assignments and opens up new avenues to using this methodology in protein structural studies.

Structural, optical and EPR studies on ZnO:Cu nanopowders prepared via low temperature solution combustion synthesis

Cu (0.1 mol%) doped ZnO nanopowders have been successfully synthesized by a wet chemical method at a relatively low temperature (300 degrees C). Powder X-ray diffraction (PXRD) analysis, scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transformed infrared (FTIR) spectroscopy, UV-Visible spectroscopy, Photoluminescence (PL) and Electron Paramagnetic Resonance (EPR) measurements were used for characterization. PXRD results confirm that the nanopowders exhibit hexagonal wurtzite structure of ZnO without any secondary phase. The particle size of as-formed product has been calculated by Williamson-Hall (W-H) plots and Scherrer's formula is found to be in the range of similar to 40 nm. TEM image confirms the nano size crystalline nature of Cu doped ZnO. SEM micrographs of undoped and Cu doped ZnO show highly porous with large voids. UV-Vis spectrum showed a red shift in the absorption edge in Cu doped ZnO. PL spectra show prominent peaks corresponding to near band edge UV emission and defect related green emission in the visible region at room temperature and their possible mechanisms have been discussed. The EPR spectrum exhibits a broad resonance signal at g similar to 2.049, and two narrow resonances one at g similar to 1.990 and other at g similar to 1.950. The broad resonance signal at g similar to 2.049 is a characteristic of Cu2+ ion whereas the signal at g similar to 1.990 and g similar to 1.950 can be attributed to ionized oxygen vacancies and shallow donors respectively. The spin concentration (N) and paramagnetic susceptibility (X) have been evaluated and discussed. (C) 2011 Elsevier B. V. All rights reserved.

Application of selective F-1 decoupled H-1 NMR for enantiomer resolution and accurate measurement of enantiomeric excess at low chiral substrate/auxiliary concentration

The higher substrate and chiral auxiliary concentration is a pre-requisite to obtain efficient separation of H-1 NMR signals of enantiomers. The higher concentration of chiral lanthanide shift reagents provides broadened spectral lines resulting in a severe loss of resolution between the enantiomer resonances. In order to circumvent such difficulties, herein we present the application and the usefulness of a selective F-1 decoupled correlation (COSY) experiment which yields proton decoupled proton spectra in the indirect dimension. The potentiality of the experiment is demonstrated on several chiral compounds possessing different functional groups, employing either a lanthanide shift reagent or a solvating reagent as chiral auxiliaries. (C) 2011 Elsevier B.V. All rights reserved.