Thermodynamics and Kinetics of Single-Chain Monellin Folding with Structural Insights into Specific Collapse in the Denatured State Ensemble

Proteins, which behave as random coils in high denaturant concentrations undergo collapse transition similar to polymers on denaturant dilution. We study collapse in the denatured ensemble of single-chain monellin (MNEI) using a coarse-grained protein model and molecular dynamics simulations. The model is validated by quantitatively comparing the computed guanidinium chloride and pH-dependent thermodynamic properties of MNEI folding with the experiments. The computed properties such as the fraction of the protein in the folded state and radius of gyration (R_g) as function of [GuHCl] are in good agreement with the experiments. The folded state of MNEI is destabilized with an increase in pH due to the deprotonation of the residues Glu24 and Cys42. On decreasing [GuHCl], the protein in the unfolded ensemble showed specific compaction. The R_g of the protein decreased steadily with [GuHCl] dilution due to increase in the number of native contacts in all the secondary structural elements present in the protein. MNEI folding kinetics is complex with multiple folding pathways and transiently stable intermediates are populated in these pathways. In strong stabilizing conditions, the protein in the unfolded ensemble showed transition to a more compact unfolded state where R_g decreased by ≈ 17% due to the formation of specific native contacts in the protein. The intermediate populated in the dominant MNEI folding pathway satisfies the structural features of the dry molten globule inferred from experiments.     

Conformational properties of the unfolded state of Im7 in nondenaturing conditions

The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, ¹⁵N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (R_h = 25 Å) relative to the urea-denatured state (R_h ≥ 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state.     

Novel bioactive molecules from Lentzea violacea strain AS 08 using one strain-many compounds (OSMAC) approach

A new eudesmane sesquiterpenoid (1), and a new homologue of virginiae butanolide E (2) along with butyl isobutyl phthalate (3) were isolated from, actinomycete-Lentzea violacea strain AS08 isolated from north western Himalayas by stressing on modified one strain-many compounds (OSMAC) method. The structures of the new compounds were elucidated by extensive spectroscopic analyses including 1D, 2D NMR along with HR-ESI-MS and FT-IR data. Herein, a distinctive method was added for inspecting secretory profile of the strain by quantification of extract value of cell free supernatant in different types of culture media fallowed by HPLC profiling of respective extracts, which revealed a highly altered metabolic profile of the strain and formed the base for the selection of media. The compounds 1 and 2 showed moderate activity against Gram negative (MIC ～32–64 µg ml^?1) in comparison to Gram positive bacterial pathogens. Compound 1 exhibited significant activity in human cancerous cell lines (IC₅₀ ～19.2 µM).     

Estimation of gross primary production of irrigated maize using Landsat-8 imagery and Eddy Covariance data

A study was conducted to understand the potential of Landsat-8 in the estimation of gross primary production (GPP) and to quantify the productivity of maize crop cultivated under hyper-arid conditions of Saudi Arabia. The GPP of maize crop was estimated by using the Vegetation Photosynthesis Model (VPM) utilizing remote sensing data from Landsat-8 reflectance (GPP_VPM) as well as the meteorological data provided by Eddy Covariance (EC) system (GPP_EC), for the period from August to November 2015. Results revealed that the cumulative GPP_EC for the entire growth period of maize crop was 1871 g C m⁻². However, the cumulative GPP determined as a function of the enhanced vegetation index – EVI (GPP_EVI) was 1979 g C m⁻², and that determined as a function of the normalized difference vegetation index – NDVI (GPP_NDVI) was 1754 g C m⁻². These results indicated that the GPP_EVI was significantly higher than the GPP_EC (R² = 0.96, P = 0.0241 and RMSE = 12.6%). While, the GPP_NDVI was significantly lower than the GPP_EC (R² = 0.93, P = 0.0384 and RMSE = 19.7%). However, the recorded relative error between the GPP_EC and both the GPP_EVI and the GPP_NDVI was −6.22% and 5.76%, respectively. These results demonstrated the potential of the landsat-8 driven VPM model for the estimation of GPP, which is relevant to the productivity and carbon fluxes.     

Synthesis,NMR characterization and divergent biological actions of 2′-hydroxy-ceramide/dihydroceramide stereoisomers in MCF7 cells

A straightforward method for the simultaneous preparation of (2S,3R,2′R)- and (2S,3R,2′S)-2′-hydroxy-ceramides (2′-OHCer) from (2S,3R)-sphingosine acetonide precursors and racemic mixtures of 2-hydroxy fatty acids (2-OHFAs) is described. The obtained 2′-OH-C4-, -C6-, -C12-, -C16-Cer and 2′-OH-C6-dhCer pairs of diastereoisomers were characterized thoroughly by TLC, MS, NMR, and optical rotation. Dynamic and multidimensional NMR studies provided evidence that polar interfaces of 2′-OHCers are extended and more rigid than observed for the corresponding non-hydroxylated analogs. Stereospecific profile on growth suppression of MCF7 cells was observed for (2′R)- and (2′S)-2′-OH-C6-Cers and their dihydro analogs. The (2′R)-isomers were more active than the (2′S)-isomers (IC₅₀ ～3 μM/8 μM and IC₅₀ ～8 μM/12 μM, respectively), surpassing activity of the ordinary C6-Cer (IC₅₀ ～12 μM) and C6-dhCer (IC₅₀ ～38 μM). Neither isomer of 2′-OH-C6-Cers and 2′-OH-C6-dhCers was metabolized to their cellular long chain 2′-OH-homologs. Surprisingly, the most active (2′R)-isomers did not influence the levels of the cellular Cers nor dhCers. Contrary to this, the (2′S)-isomers generated cellular Cers and dhCers efficiently. In comparison, the ordinary C6-Cer and C6-dhCer also significantly increased the levels of their cellular long chain homologs. These peculiar anabolic responses and SAR data suggest that (2′R)-2′-OHCers/dhCers may interact with some distinct cellular regulatory targets in a specific and more effective manner than their non-hydroxylated analogs. Thus, stereoisomers of 2′-OHCers can be potentially utilized as novel molecular tools to study lipid–protein interactions, cell signaling phenomena and to understand the role of hydroxylated sphingolipids in cancer biology, pathogenesis and therapy.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Phytochemical and cytotoxic studies on the leaves of Calotropis gigantea

A new lignan, 9′-methoxypinoresinol (1), and two new glycosylated 5-hydroxymethylfurfurals, calofurfuralside A (2), and calofurfuralside B (3), together with nine known compounds (4–12) have been isolated from the active fractions, CHCl₃ (IC₅₀, 0.32 μg mL^?1) and EtOAc (IC₅₀, 0.55 μg mL^?1) fractions of the leaves of Calotropis gigantea. Their structures were elucidated based on NMR and MS data. Among the isolated compounds, compounds 1 and 9 exhibited potent cytotoxicity against PANC-1 human pancreatic cancer cell line under the normoglycemic condition with IC₅₀ values of 3.7 and 3.3 μM, respectively. 9′-Methoxypinoresinol (1) significantly inhibited the colony formation of PANC-1 cells in a concentration-dependent manner.     

Facile purification of mono-PEGylated interleukin-1 receptor antagonist and its characterization with multi-angle laser light scattering

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was chemically conjugated with succinimidyl carbonate monomethoxyl polyethylene glycols of 5 kDa (SC-PEG_5k) and 10 kDa (SC-PEG_10k) molecular weight. A facile purification of the conjugates was achieved by one-step cationic exchange chromatography. The purity of mono-PEGylated protein was greater than 95%. The purified conjugate was characterized by multi-angle laser light scattering (MALLS) for determining the apparent gyration radius (r_g) and hydrodynamic radius (r_h). MALLS results showed that the conjugation of PEG markedly enhanced r_g and r_h of parent protein (r_g: from 15.7 to 48.2 nm for the PEG_5k and 81.9 nm for the PEG_10k; r_h: from 4.2 to 58.4 nm for the PEG_5k and 102.3 nm for the PEG_10k). The PEGylated rhIL-1ra retained 44.6% of binding activities to the cell receptor for PEG_5k and 40.2% for PEG_10k, compared to the original protein.     

Design,synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1′-P2′ ligands

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1′ and P2′ ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (K_i = 2 nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23 nM and cellular EC₅₀ of 80 nM.     

Morphology and bloom dynamics of Cochlodinium geminatum (Schütt) Schütt in the Pearl River Estuary,South China Sea

An unarmored dinoflagellate bloom of Cochlodinium geminatum (Schütt) Schütt has been identified in the Pearl River Estuary, South China Sea during the severe dry season, from late October to early November, 2009, when temperature and salinity ranged between 20.0–27.2 °C and 10.6–33.4, respectively. Light and scanning electron microscopy were used to identify the characteristics of C. geminatum and provided the clear morphological structure for this species. The organism was primarily found in chains of two cells or single cell, and no longer chains were observed. Cells were irregularly spherical or slightly dorso-ventrally, with size ranged between 28 and 36 μm and longer than wide. A large nucleus in the center with numerous golden chloroplasts was present, and the cingulum made 1.5 turns around the cell. The concentration of C. geminatum ranged from 10² to greater than 10⁷ cells l⁻¹ during the bloom period. Nutrient concentration ranges during the bloom were 1.29–81.00 μM NO₃, 0.14–12.14 μM NO₂, 0.21–6.29 μM NH₄, 0.23–6.26 μM PO₄ and 3.29–171.43 μM SiO₃, respectively. Total biomass expressed in terms of chlorophyll a ranged from 2.44 to 135.45 μg l⁻¹, with an average 19.9 μg l⁻¹ in surface water throughout the PRE. Two main clusters corresponding to the water sectors were defined with multivariate analysis (cluster and nMDS). Based on the composition and abundance of phytoplankton, spatial variations were observed at a significant level (ANOSIM, R = 0.44, P < 0.01). Although the pairwise correlation analysis detected no significant effect of any single environmental variable on the abundance of C. geminatum, the multivariate analysis (BIO-ENV) between biotic and abiotic variables resulted in the best variables combination with all measured factors involved (temperature, salinity, turbidity, NO₃, NO₂, NH₄, PO₄ and SiO₃) which showed a combined effect during the bloom of C. geminatum in the Pearl River Estuary (ρ_w = 0.477).     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).     

Synthesis and evaluation of the NSCLC anti-cancer activity and physical properties of 4-aryl-N-phenylpyrimidin-2-amines

Reported herein are efforts to profile 4-aryl-N-phenylpyrimidin-2-amines in terms of their anti-cancer activity towards non small-cell lung carcinoma (NSCLC) cells. We have synthesized new 4-aryl-N-phenylpyrimidin-2-amines and assessed them in terms of their cytotoxicity (A549, NCI-H187, MCF7, Vero & KB) and physicochemical properties (logD_7.4 and solubility). 13f and 13c demonstrated potent anti-cancer activity in A549 cells (0.2 µM), compared to 0.4 μM for the NSCLC drug Doxorubicin. 13f also displayed low experimental logD_7.4 (2.9) and the best solubility (～40 μM). Compounds 13b and 13d showed the best balance of A549 anti-cancer activity and selectivity. 13g showed good activity and selectivity comparable with the anti-cancer drug Doxorubicin.     

Traveling behavior of a termite in tunnels with different curvatures and base surface roughness

This study focused on understanding the termite traveling behavior. Artificial tunnels with different curvatures and base surface roughness were constructed. Each tunnel was 50 mm in length, with widths of W (W = 2, 3, or 4 mm). The distance between the two ends of the tunnel was D (D = 20, 30, 40, or 50 mm). A higher value of D means a lower curvature. The roughness, R (R = 60, 120, 240 and ∞), was generated by uniformly sanding the substrate with a sanding machine. A higher R value means a finer base surface. The symbol ∞ represents a smooth surface. Time (τ) taken by a termite to pass through the tunnel was measured. τ was longer in tunnels with a smooth surface compared with the tunnels with a rough surface. When W = 2, there was no effect of the roughness on speed. This is because the narrow tunnel width diluted the effects of the smoothness of the surface. When W = 3, τ was statistically shorter for R = 240, than for R = 60 and 120. This suggests that an appropriate surface roughness could positively contribute to the traveling speed.     

Effects of shell morphological traits on the weight trait of the orange strain of the Manila clam

A new strain of Manila clam with orange shell color was produced after selection within a full-sib family for two generations. In the present study, the shell length, height, and width, and the live body weight of the orange strain were measured, and their correlation coefficients were calculated. The shell morphological traits were used as independent variables, and the live body weight was used as the dependent variable for calculating the path coefficients, correlation index, and determination coefficients. The results showed that the correlation coefficients between each shell morphological trait and the live body weight were all highly significant (P < 0.01). The correlation indices (R²) of morphological traits against the live body weight of clams were larger than 0.85, indicating that the morphology traits were the main factors affecting the body weight. Multiple regression equations were obtained to estimate shell length X₁ (cm), shell height X₂ (cm), and shell width X₃ (cm) against live body weight Y (g): Y = ? 2.62 + 0.34 X₁ + 0.145 X₂, (X₁ < 0.05, X₂ < 0.05). The results suggest that the shell length could be used as the main trait for selective breeding and could indirectly make a large improvement in the weight trait.     

Antiparasitic lethality of sulfonamidebenzamides in kinetoplastids

A sulfonamidebenzamide series was assessed for anti-kinetoplastid parasite activity based on structural similarity to the antiparasitic drug, nifurtimox. Through structure-activity optimization, derivatives with limited mammalian cell toxicity and increased potency toward African trypanosomes and Leishmania promastigotes were developed. Compound 22 had the best potency against the trypanosome (EC₅₀ = 0.010 μM) while several compounds showed ～10-fold less potency against Leishmania promastigotes without impacting mammalian cells (EC₅₀ > 25 μM). While the chemotype originated from an unrelated optimization program aimed at selectively activating an apoptotic pathway in mammalian cancer cells, our preliminary results suggest that a distinct mechanism of action from that observed in mammalian cells is responsible for the promising activity observed in parasites.     

Diversity and bioactivities of nostocacean cyanobacteria isolated from paddy soil in Vietnam

Nostocacean cyanobacteria are one of the important components of paddy fields due to their ability to fix atmospheric nitrogen and supply phytohormones for crop growth. In this study, 13 Nostoc strains isolated from paddy soils in Vietnam were classified using a polyphasic approach. The results showed a high diversity of the isolated strains that represented seven morphotypes corresponding to five genotypes, with 16S rRNA gene sequence similarity values ranging between 94.97–99.78% compared to the available sequences from GenBank. Bioassay assessment revealed that 11 out of 13 strains possessed antibacterial activities, three of which exhibited cytotoxic activities on MCF7 and HCT116 cells with an IC₅₀ ranging from 47.8 μg mL^?1 to 232.0 μg mL^?1. Interestingly, strains with identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activity profiles.     

On the Calculation of SAXS Profiles of Folded and Intrinsically Disordered Proteins from Computer Simulations

Solution techniques such as small-angle X-ray scattering (SAXS) play a central role in structural studies of intrinsically disordered proteins (IDPs); yet, due to low resolution, it is generally necessary to combine SAXS with additional experimental sources of data and to use molecular simulations. Computational methods for the calculation of theoretical SAXS intensity profiles can be separated into two groups, depending on whether the solvent is modeled implicitly as continuous electron density or considered explicitly. The former offers reduced computational cost but requires the definition of a number of free parameters to account for, for example, the excess density of the solvation layer. Overfitting can thus be an issue, particularly when the structural ensemble is unknown. Here, we investigate and show how small variations of the contrast of the hydration shell, δρ, severely affect the outcome, analysis and interpretation of computed SAXS profiles for folded and disordered proteins. For both the folded and disordered proteins studied here, using a default δρ may, in some cases, result in the calculation of non-representative SAXS profiles, leading to an overestimation of their size and a misinterpretation of their structural nature. The solvation layer of the different IDP simulations also impacts their size estimates differently, depending on the protein force field used. The same is not true for the folded protein simulations, suggesting differences in the solvation of the two classes of proteins, and indicating that different force fields optimized for IDPs may cause expansion of the polypeptide chain through different physical mechanisms.     

Anthropometric dimensions provide reliable estimates of abdominal adiposity: A validation study

Abdominal fat accumulation is a major risk factor for cardiometabolic morbidity and mortality. The purpose of the study is to assess the possibility of developing accurate estimation equations based on body measurements to determine total abdominal (TFA), subcutaneous (SFA) and visceral fat area (VFA). Hungarian volunteers (n = 198) aged between 20 and 81 years were enrolled in the study, which was conducted between July and November 2014. All persons underwent anthropometric measurements and computer tomographic (CT) scanning. Sex-specific multiple linear regression analyses were conducted in a subgroup of 98 participants to generate estimation models, then Bland–Altman's analyses were applied in the cross-validation group to compare their predictive efficiency. The variables best predicting VFA were hip circumference, calf circumference and waist-to-hip ratio (WHR) for males (R² = 0.713; SEE = 5602.1 mm²) and sagittal abdominal diameter (SAD), WHR, thigh circumference and triceps skinfold for females (R² = 0.845; SEE = 3835.6 mm²). The SFA prediction equation included SAD, thigh circumference and abdominal skinfold for males (R² = 0.848; SEE = 4124.1 mm²), body mass index and thigh circumference for females (R² = 0.861; SEE = 5049.7 mm²). Prediction accuracy was the highest in the case of TFA: hip circumference and WHR for males (R² = 0.910; SEE = 5637.2 mm²), SAD, thigh circumference and abdominal skinfold for females (R² = 0.915; SEE = 6197.5 mm²) were used in the equations. The results suggested that deviations in the predictions were independent of the amount of adipose tissue. Estimation of abdominal fat depots based on anthropometric traits could provide a cheap, reliable method in epidemiologic research and public health screening to evaluate the risk of cardiometabolic events.     

Improvement of (R)-carbonyl reductase-mediated biosynthesis of (R)-1-phenyl-1,2-ethanediol by a novel dual-cosubstrate-coupled system for NADH recycling

An NAD(H)-dependent (R)-carbonyl reductase (RCR) from Candida parapsilosis catalyzes the asymmetric reduction of 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED), which is a valuable chiral building block in the pharmaceutical and fine chemical industries. Biosynthesis efficiency of (R)-PED was considerably improved by a novel dual-cosubstrate-coupled system. By simultaneously employing isopropanol (10%, v v^?1) and glycerol (8%, v v^?1) as sacrificial cosubstrates, the (R)-PED product had an excellent optical purity of >99.9% and a conversion of 85.5%, which were nearly 2- and 11-fold higher than those without adding cosubstrate, respectively. Besides, the productivity was dramatically enhanced from 0.02 g L^?1 h^?1 to 5 g L^?1 h^?1, and the maximum acceptable concentration of 2-HAP was elevated to 10 g L^?1. Isopropanol was directly oxidized by RCR in the formation of NADH, while glycerol was metabolized by cellular enzymes to release NADH. Moreover, glycerol prevented cells from losing viability and alleviated the toxicity of isopropanol and acetone for cells. Interestingly, there was a cooperative interaction between isopropanol and glycerol for the improvement of biosynthesis efficiency of (R)-PED.     

Electronic structure of [Ga2(tren)2(CAsq,cat)](BPh4)2(BF4): An EPR,ENDOR, and density functional study

The bimetallic [M₁M₂(tren)₂(CA^n?)]^m+ series, where M = Ga^III or Cr^III and CA is the chloranilate ligand which can take on diamagnetic (CA^cat,cat)^4? or paramagnetic (CA^sq,cat)^3? forms, comprises an electronically diverse series of compounds ranging from the closed-shell [Ga₂(tren)₂(CA^cat,cat)]²⁺ to the S = 5/2 ground state of [Cr₂(tren)₂(CA^sq,cat)]³⁺. This report deals with the interpretation of the EPR and ENDOR spectra of [Ga₂(tren)₂(CA^sq,cat)](BPh₄)₂(BF₄) (2) and the related derivative [Ga₂(tren)₂(DHBQ)](BPh₄)₂(BF₄) (2a) (where DHBQ is the fully deprotonated trianionic form of 2,5-dihydroxy-1,4-benzoquinone) in an effort to further characterize the electronic structure of this radical species. The X-band (～9.5 GHz) EPR spectrum of complex 2 acquired in a butyronitrile/propionitrile glass at 4 K reveals a rhombic g-tensor with g_xx = 2.0100, g_yy = 2.0097, and g_zz = 2.0060 with hyperfine interactions due to spin delocalization onto the two Ga nuclei (a_xx = 4.902 G, a_yy = 4.124 G, a_zz = 3.167 G); the origin of the hyperfine coupling was confirmed by analysis of the room temperature spectra of complexes 2 and 2a. The low-temperature spectrum of complex 2 also indicates the presence of a triplet electronic state characterized by a g-value of 2.009 and axial zero-field splitting of D = 150 G (0.012 cm^?1) as determined from measurements carried out at both X- and W-band (～95 GHz) frequencies. This triplet state is believed to arise due to a weak intermolecular Heisenberg exchange interaction between two aggregating complexes. ENDOR measurements on complex 2a at 20 K allowed for a determination of the magnitude of hyperfine coupling to the protons associated with the radical bridge as well as providing a rare example of an ENDOR signal arising from coupling to a gallium nucleus. Finally, these results were combined with literature data on the free semiquinone form of the bridging ligand in order to assess the extent to which density functional theory can predict unpaired spin density distribution in a complex molecule of this type. Although differences between theory and experiment were noted, DFT was able to provide a reasonably accurate picture of the electronic structure of this system as well as provide insight into the spin polarization mechanism(s) responsible for the observed hyperfine interactions.