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The nanoscale parameters of metal clusters and lattices have a crucial influence on the macroscopic properties of materials. Herein, we provide a detailed study on the size and shape of isolated yttrium carbide clusters in different fullerene cages. A family of diyttrium endohedral metallofullerenes with the general formula of Y(2)C(2n) (n = 40-59) are reported. The high field (13)C nuclear magnetic resonance (NMR) and density functional theory (DFT) methods are employed to examine this yttrium carbide cluster in certain family members, Y(2)C(2)@D(5)(450)-C(100), Y(2)C(2)@D(3)(85)-C(92), Y(2)C(2)@C(84), Y(2)C(2)@C(3v)(8)-C(82), and Y(2)C(2)@C(s)(6)-C(82). The results of this study suggest that decreasing the size of a fullerene cage with the same (Y(2)C(2))(4+) cluster results in nanoscale fullerene compression (NFC) from a nearly linear stretched geometry to a constrained "butterfly" structure. The (13)C NMR chemical shift and scalar (1)J(YC) coupling parameters provide a very sensitive measure of this NFC effect for the (Y(2)C(2))(4+) cluster. The crystal structural parameters of a previously reported metal carbide, Y(2)C(3) are directly compared to the (Y(2)C(2))(4+) cluster in the current metallofullerene study.

The members of a new family of yttrium trimetallic nitride-templated (TNT) endohedral metallofullerenes (EMFs), Y(3)N@C(2n) (n = 40-43), have been synthesized and purified. On the basis of experimental and computational (13)C NMR studies, we propose cage structures for Y(3)N@I(h)-C(80) (IPR allowed), Y(3)N@D(5h)-C(80) (IPR allowed), Y(3)N@C(s)-C(82) (non-IPR), Y(3)N@C(s)-C(84) (non-IPR), and Y(3)N@D(3)-C(86) (IPR allowed). A significant result is the limited number of isomers found for each carbon cage. For example, there are 24 isolated pentagon rule (IPR) and 51 568 non-IPR structures possible for the C(84) cage, but only one major isomer of Y(3)N@C(s)-C(84) was found. The current study confirms the unique role of the trimetallic nitride (M(3)N)(6+) cluster template in the Kratschmer-Huffman electric-arc process for fullerene cage size and high symmetry isomer selectivity. This study reports the first (89)Y NMR results for Y(3)N@I(h)-C(80,) Y(3)N@C(s)(51365)-C(84), and Y(3)N@D(3)(19)-C(86), which reveal a progression from isotropic to restricted (Y(3)N)(6+) cluster motional processes. Even more surprising is the sensitivity of the (89)Y NMR chemical shift parameter to subtle changes in the electronic environment at each yttrium nuclide in the (Y(3)N)(6+) cluster (more than 200 ppm for these EMFs). This (89)Y NMR study suggests that (89)Y NMR will evolve as a powerful tool for cluster motional studies of EMFs.

trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R(G)) disappeared in the spectrum of the inactive alphaR8A mutant (deltaH = 7.48 ppm, deltaN = 89.6 ppm), thereby assigning these resonances to alphaArg-11NepsilonH, and alphaArg-8NepsilonH, respectively. (1)H-(15)N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoic acid (3-CBA), a competitive inhibitor (K(I)(slope) = 0.35 +/- 0.06 mM), resulted in progressive downfield shifts of the alphaArg-8Nepsilon resonance yielding a K(D) = 0.77 +/- 0.44 mM, comparable to the (K(I)(slope), suggestive of active site binding. Increasing the pH of free CaaD to 8.9 at 5 degrees C resulted in the disappearance of all nine Arg-NepsilonH resonances due to base-catalyzed NepsilonH exchange. Saturating the enzyme with 3-CBA (16 mM) induced the reappearance of two NepsilonH signals, those of alphaArg-8 and alphaArg-11, indicating that the binding of the substrate analogue 3-CBA selectively slows the NepsilonH exchange rates of these two arginine residues. The kinetic and NMR data thus indicate that betaPro-1 is the acid catalyst, alphaGlu-52 is a reasonable c...

Edited by Gerald W. HartAvian coronaviruses, including infectious bronchitis virus (IBV), are important respiratory pathogens of poultry. The heavily glycosylated IBV spike protein is responsible for binding to host tissues. Glycosylation sites in the spike protein are highly conserved across viral genotypes, suggesting an important role for this modification in the virus life cycle. Here, we analyzed the N-glycosylation of the receptor-binding domain (RBD) of IBV strain M41 spike protein and assessed the role of this modification in host receptor binding. Ten single Asn-to-Ala substitutions at the predicted N-glycosylation sites of the M41-RBD were evaluated along with two control Val-to-Ala substitutions. CD analysis revealed that the secondary structure of all variants was retained compared with the unmodified M41-RBD construct. Six of the 10 glycosylation variants lost binding to chicken trachea tissue and an ELISA-presented ␣2,3-linked sialic acid oligosaccharide ligand. LC/MS E glycomics analysis revealed that glycosylation sites have specific proportions of N-glycan subtypes. Overall, the glycosylation patterns of most variant RBDs were highly similar to those of the unmodified M41-RBD construct. In silico docking experiments with the recently published cryo-EM structure of the M41 IBV spike protein and our glycosylation results revealed a potential ligand receptor site that is ringed by four glycosylation sites that dramatically impact ligand binding. Combined with the results of previous array studies, the glycosylation and mutational analyses presented here suggest a unique glycosylation-dependent binding modality for the M41 spike protein.

The conformations of two synthetic trisaccharides of blood group A and B (a-L-Fucp- (1 2were studied by NMR measurements of one-bond C H residual dipolar couplings in partially oriented liquid crystal solutions. The conformations of the three oligosaccharides were analyzed by generating thousands of structures using a Monte-Carlo method. Two different strategies were applied to calculate theoretical dipolar couplings for these structures. In the first method, the orientation of the molecule was calculated from the optimal fit of the molecular model to the experimental data, while in the second method the orientation tensor was calculated directly from the moment of inertia of the molecular model. Both methods of analysis give similar results but with slightly better agreement with experiment for the former one. The analysis of the results implies a single unique conformation for both blood group epitopes in solution in disagreement with theoretical models suggesting the existence of two conformers in solution.

Oomycetes, such as Phytophthora sojae, are plant pathogens that employ protein effectors that enter host cells to facilitate infection. Plants may overcome infection by recognizing pathogen effectors via intracellular receptors (R proteins) that form part of their defense system. Entry of some effector proteins into plant cells is mediated by conserved RxLR motifs in the effectors and phosphoinositides (PIPs) resident in the host plasma membrane such as phosphatidylinositol 3-phosphate (PtdIns(3)P). Recent reports differ regarding the regions on RxLR effector proteins involved in PIP recognition. To clarify these differences, I have structurally and functionally characterized the P. sojae effector, avirulence homolog-5 (Avh5).

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