Solution NMR Structure and Backbone Dynamics of Partially Disordered <i>Arabidopsis thaliana</i> Phloem Protein 16-1, a Putative mRNA Transporter

Although RNA-binding proteins in plant phloem are believed to perform long-distance systemic transport of RNA in the phloem conduit, the structure of none of them is known. <i>Arabidopsis thaliana</i> phloem protein 16-1 (<i>At</i>PP16-1) is such a putative mRNA transporter whose structure and backbone dynamics have been studied at pH 4.1 and 25 °C by high-resolution nuclear magnetic resonance spectroscopy. Results obtained using basic optical spectroscopic tools show that the protein is unstable with little secondary structure near the physiological pH of the phloem sap. Fluorescence-monitored titrations reveal that <i>At</i>PP16-1 binds not only <i>A</i>. <i>thaliana</i> RNA (<i>K</i>_diss ∼ 67 nM) but also sheared DNA and model dodecamer DNA, though the affinity for DNA is ∼15-fold lower. In the solution structure of the protein, secondary structural elements are formed by residues 3–9 (β1), 56–62 (β2), 133–135 (β3), and 96–110 (α-helix). Most of the rest of the chain segments are disordered. The N-terminally disordered regions (residues 10–55) form a small lobe, which conjoins the rest of the molecule via a deep and large irregular cleft that could have functional implications. The average order parameter extracted by model-free analysis of ¹⁵N relaxation and {¹H}–¹⁵N heteronuclear NOE data is 0.66, suggesting less restricted backbone motion. The average conformational entropy of the backbone NH vectors is −0.31 cal mol^–1 K^–1. These results also suggest structural disorder in <i>At</i>PP16-1

Sequential Allylic Substitution/Pauson–Khand Reaction: A Strategy to Bicyclic Fused Cyclopentenones from MBH-Acetates of Acetylenic Aldehydes

An efficient approach for the construction of novel bicyclic fused cyclopentenones starting from Morita–Baylis–Hillman (MBH) acetates of acetylenic aldehydes with flexible scaffold diversity has been achieved using a two-step reaction sequence involving allylic substitution and the Pauson–Khand reaction. This strategy provided a facile access to various bicyclic cyclopentenones fused with either a carbocyclic or a heterocyclic ring system in good yield

Synthesis of Proposed Aglycone of Mandelalide A

A highly convergent synthesis of the proposed mandelalide A aglycone is reported. The cornerstones of the synthetic strategy include the following: <i>E</i>-selective intramolecular Heck cyclization, Masamune–Roush olefination, Stork–Zhao–Wittig olefination, modified Prins cyclization; Sharpless asymmetric dihydroxylation followed by Williamson-type etherification, Julia–Kocienski olefination, Brown crotylation, and Brown allylation reactions

Conventional steps in protein structure determination by solution state NMR spectroscopy.

<p>Conventional steps in protein structure determination by solution state NMR spectroscopy.</p

Overview of ADAPT NMR. The shaded rectangle at the left shows the sample and operations carried out by the NMR spectrometer.

<p>Initial input is indicated in the upper left corner. The dashed line encloses components of the probabilistic network. The ADAPT-NMR output is listed at the right.</p

Dynamics of apo-CobY as Represented by Residue-specific Backbone Amide Spin-lattice (<i>T</i>₁) and Spin-spin (<i>T</i>₂) Relaxation Times, Heteronuclear NOE (¹⁵N NOE) Values, and Correlation Times.

<p>The flexible regions of the polypeptide chain are circled in red; the vertical red box represents missing resonances from residues that are assumed to be flexible; and the horizontal red box indicates the C-terminal helix, which appears to be more flexible than other secondary features as indicated by the smaller ¹⁵N NOE values. The τ_c values were calculated from measured <i>T</i>₁ and <i>T</i>₂ values by using the formula, τ_c = 1/4πν_N (√6(<i>T</i>1/<i>T</i>2) − 7). The average τ_c value (9.9 ± 0.7 ns) indicates that CobY is monomeric in solution under the NMR sample conditions.</p

Results from ADAPT-NMR data collection and backbone analysis of six proteins.

a<p>This work.</p

Overlay of the Ribbon Structure apo-CobY (cyan) with those of Structurally Similar Proteins.

<p>(A) (green) X-ray structure of the CobY^G153D:GTP complex (PDB 3RSB) (87% structural overlap with rmsd 1.82 Å; fragment/topology score 0.95/0.94 with match size 113). (B) (gray) X-ray structure of CMP:2-keto-3-deoxy-manno-octonic acid synthetase (PDB 1H7F).(75.5% structural overlap with rmsd 1.84 Å; fragment/topology score 0.93/1.0 with match size 148). (C) (gray) X-ray structure of α-D-glucose-1-phosphate cytidylyl transferase (PDB 1WVC) (75.5% structural overlap with rmsd 2.15 Å; fragment/topology score 0.86/1.0 with match size 148). (D) (gray) X-ray structure of acytidylyl transferase (PDB 2VSI) (74.5% structural overlap with rmsd 1.98 Å; fragment/topology score 0.91/1.0 with match size 146). (E) X-ray structure of 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase(PDB 1VPA) (73% structural overlap with rmsd 1.86 Å; fragment/topology score 0.91/1.0 with match size 143). (F) X-ray structure of CMP-acylneuraminate synthetase (PDB 1EYR) (70% structural overlap with rmsd 1.95 Å; fragment/topology score 0.88/1.0 with match size 137). PYMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref020" target="_blank">20</a>] was used to generate the structures, and the CLICK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref033" target="_blank">33</a>] program was used to carry out the pairwise alignments.</p

Comparison of Structures of apo-CobY and CobY^G153D:GTP Complex and Comparison of ¹H-¹⁵N HSQC Spectra of CobY and CobY:GTP.

<p>(A) Superposition of the 3D structures of apo-CobY determined by NMR (cyan) and CobY^G153D:GTP determined by X-ray crystallography (green) with the GTP displayed as a stick model. (B) Expansion of the overlaid structures showing the GTP binding site. Residues perturbed upon complex formation with GTP are annotated. Whereas α-helix-III (circled in red) is well defined in the structure of apo-CobY, it is poorly resolved in the X-ray structure of the complex. (C) Weighted rmsd chemical shift differences ([0.5[Δδ(¹H^N)²+ (0.2Δδ(¹⁵N))²]]^1/2) between apo-CobY and CobY:GTP mapped onto the X-ray structure of CobY^G153D:GTP. The magnitude of the shift is coded by a spectrum with red largest shift and blue small or no shift. Residues not detected or assigned in both NMR in the spectra compared are shown in gray. The dotted regions of the polypeptide chain represent ones for which no electron density was detected (D-F) Overlays of three regions of the ¹⁵N HSQC spectra of CobY (blue) and CobY:GTP (red). The cross peaks from CobY are annotated with their assignments, and the arrows indicate the changes in peak positions upon complex formation with GTP.</p

Three-dimensional Solution Structure of apo-free CobY.

<p>(A) Secondary structure topology representing seven β-stands and five α-helices. (B) Ribbon diagram of the lowest energy conformer of apo-CobY. (C) Overlay of the regular secondary structure of the 20 lowest energy conformers that showed the fewest violations in CYANA (N- and C-terminals are labeled). (D) The rmsd of the backbone atoms plotted against the corresponding amino acid number. Residues 10–20 and 135–150 displayed the highest rmsd values. Also shown are the positions of the α-helices and β-strands.</p