Long-range heteronuclear J-coupling constants in esters: Implications for 13C metabolic MRI by side-arm parahydrogen-induced polarization

Side-arm parahydrogen induced polarization (PHIP-SAH) presents a cost-effective method for hyperpolarization of 13C metabolites (e.g. acetate, pyruvate) for metabolic MRI. The timing and efficiency of typical spin order transfer methods including magnetic field cycling and tailored RF pulse sequences crucially depends on the heteronuclear J coupling network between nascent parahydrogen protons and 13C, post-parahydrogenation of the target compound. In this work, heteronuclear nJHC (1 < n ≤ 5) couplings of acetate and pyruvate esters pertinent for PHIP-SAH were investigated experimentally using selective HSQMBC-based pulse sequences and numerically using DFT simulations. The CLIP-HSQMBC technique was used to quantify 2/3-bond JHC couplings, and 4/5-bond JHC ≲ 0.5 Hz were estimated by the sel-HSQMBC-TOCSY approach. Experimental and numerical (DFT-simulated) nJHC couplings were strongly correlated (P < 0.001). Implications for 13C hyperpolarization by magnetic field cycling, and PH-INEPT and ESOTHERIC type spin order transfer methods for PHIP-SAH were assessed, and the influence of direct nascent parahydrogen proton to 13C coupling when compared with indirect homonuclear TOCSY-type transfer through intermediate (non-nascent parahydrogen) protons was studied by the density matrix approach

Characterization of wound responsive genes in Aquilaria malaccensis.

We report on the isolation and characterization of several genes responsive to wounding in the tropical endangered tree Aquilaria malaccensis. Wounding triggers the formation of a fragrant substance inside the tree stem. Deduced amino acid of the cloned sequences exhibited sequence similarities to their respective homologs: transcription factors of the WRKY gene family (AmWRKY) and β-1,3-glucanase (AmGLU). A homolog to phenylalanine ammonia-lyase (AmPAL) from previous work was also included. All cDNA sequences were of partial lengths. We studied their expression profiles in a wounding-stress experiment. Mechanical wounding induces AmWRKY in an early response to wounding (3 h), and elevates AmPAL and AmGLU expressions after 16 h. It is possible that AmWRKY mediates early wounding response while AmPAL mediates response to fungal infection by co-inducing AmGLU. Their homologs in other plants are known to inhibit fungal growth. Our data provide the first insight into the mechanisms of wounding responses in Aquilaria

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization

P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5. © 2021 Elsevier Ltd1

Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme

In this article, we report the total chemical synthesis of human lysozyme. Lysozyme serves as a widespread model system in various fields of biochemical research, including protein folding, enzyme catalysis, and amyloidogenesis. The 130-aa wild-type polypeptide chain of the human enzyme was assembled from four polypeptide segments by using native chemical ligation in a fully convergent fashion. Key to the assembly strategy is the application of the recently developed kinetically controlled ligation methodology, which provides efficient control over the ligation of two peptide αthioesters to yield a unique product. This result enables the facile preparation of a 64-residue peptide αthioester; this segment is joined by native chemical ligation to a 66-aa Cys peptide, to yield the target 130-aa polypeptide chain. The synthetic polypeptide chain was folded in vitro into a defined tertiary structure with concomitant formation of four disulfides, as shown by 2D TOCSY NMR spectroscopy. The structure of the synthetic human lysozyme was confirmed by high-resolution x-ray diffraction, giving the highest-resolution structure (1.04 Å) observed to date for this enzyme. Synthetic lysozyme was obtained in good yield and excellent purity and had full enzymatic activity. This facile and efficient convergent synthesis scheme will enable preparation of unique chemical analogs of the lysozyme molecule and will prove useful in numerous areas of lysozyme research in the future