Roots-eye view: using microdialysis and microCT to non-destructively map root nutrient depletion and accumulation zones

Improvement in fertiliser use efficiency is a key aspect for achieving sustainable agriculture in order to minimise costs, greenhouse gas emissions and pollution from nutrient runoff. To optimise root architecture for nutrient uptake and efficiency we need to understand what the roots encounter in their environment. Traditional methods of nutrient sampling such as salt extractions can only be done at the end of an experiment, are impractical for sampling locations precisely and give total nutrient values which can overestimate the nutrients available to the roots. In contrast, microdialysis provides a non-invasive, continuous method for sampling available nutrients in the soil. Here for the first time we have used microCT imaging to position microdialysis probes at known distances from the roots and then measured the available nitrate and ammonium. We found that nitrate accumulated close to roots while ammonium was depleted demonstrating that this combination of complementary techniques provides a unique ability to measure root-available nutrients non-destructively and in almost real-time

Structure-Correlation NMR Spectroscopy for Macromolecules Using Repeated Bidirectional Photoisomerization of Azobenzene

Control over macromolecular structure offers bright potentials for manipulation of macromolecular functions. We here present structure-correlation NMR spectroscopy to analyze the correlation between polymorphic macromolecular structures driven by photoisomerization of azobenzene. The structural conversion of azobenzene was induced within the mixing time of a NOESY experiment using a colored light source, and the reverse structural conversion was induced during the relaxation delay using a light source of another color. The correlation spectrum between <i>trans</i>- and <i>cis</i>-azobenzene was then obtained. To maximize the efficiency of the bidirectional photoisomerization of azobenzene-containing macromolecules, we developed a novel light-irradiation NMR sample tube and method for irradiating target molecules in an NMR radio frequency (rf) coil. When this sample tube was used for photoisomerization of an azobenzene derivative at a concentration of 0.2 mM, data collection with reasonable sensitivity applicable to macromolecules was achieved. We performed isomerization of an azobenzene-cross-linked peptide within the mixing time of a NOESY experiment that produced cross-peaks between helix and random-coil forms of the peptide. Thus, these results indicate that macromolecular structure manipulation can be incorporated into an NMR pulse sequence using an azobenzene derivative and irradiation with light of two types of wavelengths, providing a new method for structural analysis of metastable states of macromolecules

Probing the Non-Native H Helix Translocation in Apomyoglobin Folding Intermediates

Apomyoglobin folds via sequential helical intermediates that are formed by rapid collapse of the A, B, G, and H helix regions. An equilibrium molten globule with a similar structure is formed near pH 4. Previous studies suggested that the folding intermediates are kinetically trapped states in which folding is impeded by non-native packing of the G and H helices. Fluorescence spectra of mutant proteins in which cysteine residues were introduced at several positions in the G and H helices show differential quenching of W14 fluorescence, providing direct evidence of translocation of the H helix relative to helices A and G in both the kinetic and equilibrium intermediates. Förster resonance energy transfer measurements show that a 5-({2-[(acetyl)­amino]­ethyl}­amino)­naphthalene-1-sulfonic acid acceptor coupled to K140C (helix H) is closer to Trp14 (helix A) in the equilibrium molten globule than in the native state, by a distance that is consistent with sliding of the H helix in an N-terminal direction by approximately one helical turn. Formation of an S108C–L135C disulfide prevents H helix translocation in the equilibrium molten globule by locking the G and H helices into their native register. By enforcing nativelike packing of the A, G, and H helices, the disulfide resolves local energetic frustration and facilitates transient docking of the E helix region onto the hydrophobic core but has only a small effect on the refolding rate. The apomyoglobin folding landscape is highly rugged, with several energetic bottlenecks that frustrate folding; relief of any one of the major identified bottlenecks is insufficient to speed progression to the transition state

Non-Native α‑Helices in the Initial Folding Intermediate Facilitate the Ordered Assembly of the β‑Barrel in β‑Lactoglobulin

The roles of non-native α-helices frequently observed in the initial folding stage of β-sheet proteins have been examined for many years. We herein investigated the residue-level structures of several mutants of bovine β-lactoglobulin (βLG) in quenched-flow pH-pulse labeling experiments. βLG assumes a collapsed intermediate with a non-native α-helical structure (I₀) in the early stage of folding, although its native form is predominantly composed of β-structures. The protection profile in I₀ of pseudo-wild type (WT*) βLG was found to deviate from the pattern of the “average area buried upon folding” (AABUF). In particular, the level of protection at the region of strand A, at which non-native α-helices form in the I₀ state, was significantly low compared to AABUF. G17E, the mutant with an increased helical propensity, showed a similar protection pattern. In contrast, the protection pattern for I₀ of E44L, the mutant with an increased β-sheet propensity, was distinct from that of WT* and resembled the AABUF pattern. Transverse relaxation measurements demonstrated that the positions of the residual structures in the unfolded states of these mutants were consistent with those of the protected residues in the respective I₀ states. On the basis of the slower conversion of I₀ to the native state for E44L to that for WT*, non-native α-helices facilitate the ordered assembly of the β-barrel by preventing interactions that trap folding

Thermodynamic parameters for the urea denaturation of p17.

<p>Thermodynamic parameters for the urea denaturation of p17.</p

The differences in the chemical shifts between E12A and WT at pH 7.

<p>The differences in the chemical shifts of (A) CA, (B) CB, (C) CO, (D) H, and (E) N are indicated. Some residue numbers are shown. The values for residue 12 were out of the shown range and were -3.2 (A) and -9.9 (B), respectively. The value for residue 9 was -0.8 (D). The locations of the helices are shown at the top of each figure with black bars for helices 2, 3, and 5, and red bars for helices 1 and 4, based on the structure of p17 (PDB: 2H3F). The values of chemical shifts are shown for E12A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167176#pone.0167176.s005" target="_blank">S1 Table</a>).</p

NMR structure of p17 (PDB: 2H3F).

<p>Red color: α1–2 loop, helices 3 and 5. The side chains for E12 and H89 are shown in blue. This figure was prepared using MOLMOL[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167176#pone.0167176.ref062" target="_blank">62</a>].</p