A Novel Tri-Enzyme System in Combination with Laser-Driven NMR Enables Efficient Nuclear Polarization of Biomolecules in Solution

NMR is an extremely powerful, yet insensitive technique. Many available nuclear polarization methods that address sensitivity are not directly applicable to low-concentration biomolecules in liquids and are often too invasive. Photochemically induced dynamic nuclear polarization (photo-CIDNP) is no exception. It needs high-power laser irradiation, which often leads to sample degradation, and photosensitizer reduction. Here, we introduce a novel tri-enzyme system that significantly overcomes the above challenges, rendering photo-CIDNP a practically applicable technique for NMR sensitivity enhancement in solution. The specificity of the nitrate reductase (NR) enzyme is exploited to selectively <i>in situ</i> reoxidize the reduced photo-CIDNP dye FMNH₂. At the same time, the oxygen-scavenging ability of glucose oxidase (GO) and catalase (CAT) is synergistically employed to prevent sample photodegradation. The resulting tri-enzyme system (NR-GO-CAT) enables prolonged sensitivity-enhanced data collection in 1D and 2D heteronuclear NMR, leading to the highest photo-CIDNP sensitivity enhancement (48-fold relative to SE-HSQC) achieved to date for amino acids and polypeptides in solution. NR-GO-CAT extends the concentration limit of photo-CIDNP NMR down to the low micromolar range. In addition, sensitivity (relative to the reference SE-HSQC) is found to be inversely proportional to sample concentration, paving the way for the future analysis of even more diluted samples

source codes for spatiotemporal kriging

Code and subroutines for data collection, Kriging, and post-processing

JMA data from 1964 to 2022

Japan Meteorological Agency (JMA) Research Vessels (OMMORV) data separated by year and saved in formats such as '.E'. There are various observation items in addition to nutrient data

Spatiotemporal Estimated Nutrients

The nutrient data reconstructed in grid format using Spatiotemporal Kriging. It is stored in '.csv' and '.RData' formats, and includes the validation results of the observation data using 10-fold cross-validation

GLODAPv2.2022 product

To verify the nutrient data estimated by STK, we provide the GLODAPv2.2022 data, which was downloaded from https://www.ncei.noaa.gov/data/oceans/ncei/ocads/data/0257247/ on September 13, 2023

NIFS data from 1964 to 2021

National Institute of Fisheries Science (NIFS) Serial Oceanographic Observation (SOO) data from Korea, separated by year and stored in '.csv' forma

preprocessed data

Preprocessed observed nutrient, ETOPO depth, and coastline data(spatial polygons), saved in binary format '.RData' for direct use in R programming language

Nuclear Magnetic Resonance Observation of α‑Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains

The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson’s disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein’s Lys C^ε amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward <i>N</i>-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS–bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys C^ε amino groups by αS–bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments

Quantitative Residue-Specific Protein Backbone Torsion Angle Dynamics from Concerted Measurement of ³<i>J</i> Couplings

Three-bond ³<i>J</i>_C′C′ and ³<i>J</i>_HNHα couplings in peptides and proteins are functions of the intervening backbone torsion angle ϕ. In well-ordered regions, ³<i>J</i>_HNHα is tightly correlated with ³<i>J</i>_C′C′, but the presence of large ϕ angle fluctuations differentially affects the two types of couplings. Assuming the ϕ angles follow a Gaussian distribution, the width of this distribution can be extracted from ³<i>J</i>_C′C′ and ³<i>J</i>_HNHα, as demonstrated for the folded proteins ubiquitin and GB3. In intrinsically disordered proteins, slow transverse relaxation permits measurement of ³<i>J</i>_C′C′ and ³<i>J</i>_HNH couplings at very high precision, and impact of factors other than the intervening torsion angle on ³<i>J</i> will be minimal, making these couplings exceptionally valuable structural reporters. Analysis of α-synuclein yields rather homogeneous widths of 69 ± 6° for the ϕ angle distributions and ³<i>J</i>_C′C′ values that agree well with those of a recent maximum entropy analysis of chemical shifts, <i>J</i> couplings, and ¹H–¹H NOEs. Data are consistent with a modest (≤30%) population of the polyproline II region

Light micrograph of diatoms isolated in this study belonging to <i>Entomoneis</i> and <i>Petodictyon</i>.

<p>(a, b) <i>Entomoneis paludosa</i> TA208. (c) <i>Entomoneis</i> sp.2 SH373. (d, e) <i>Entomoneis</i> sp.3 EW239. (f, g) <i>Entomoneis</i> sp.1 TA410. (h) <i>Petrodictyon gemma</i> TA201. Scale bar = 10 μm.</p