Oxygenic phototrophs perform carbon fixation through the Calvin–Benson cycle. Different mechanisms adjust the cycle and the light‐harvesting reactions to rapid environmental changes. Photosynthetic ...glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) is a key enzyme in the cycle. In land plants, different photosynthetic GAPDHs exist: the most abundant isoform is formed by A2B2 heterotetramers and the least abundant by A4 homotetramers. Regardless of the subunit composition, GAPDH is the major consumer of photosynthetic NADPH and its activity is strictly regulated. While A4‐GAPDH is regulated by CP12, AB‐GAPDH is autonomously regulated through the C‐terminal extension (CTE) of its B subunits. Reversible inhibition of AB‐GAPDH occurs via the oxidation of a cysteine pair located in the CTE and the substitution of NADP(H) with NAD(H) in the cofactor‐binding site. These combined conditions lead to a change in the oligomerization state and enzyme inhibition. SEC–SAXS and single‐particle cryo‐EM analysis were applied to reveal the structural basis of this regulatory mechanism. Both approaches revealed that spinach (A2B2)n‐GAPDH oligomers with n = 1, 2, 4 and 5 co‐exist in a dynamic system. B subunits mediate the contacts between adjacent tetramers in A4B4 and A8B8 oligomers. The CTE of each B subunit penetrates into the active site of a B subunit of the adjacent tetramer, which in turn moves its CTE in the opposite direction, effectively preventing the binding of the substrate 1,3‐bisphosphoglycerate in the B subunits. The whole mechanism is made possible, and eventually controlled, by pyridine nucleotides. In fact, NAD(H), by removing NADP(H) from A subunits, allows the entrance of the CTE into the active site of the B subunit, hence stabilizing inhibited oligomers.
Regulation of the heteromeric form of photosynthetic glyceraldehyde 3‐phosphate dehydrogenase (AB‐GAPDH) depends on oscillation between a fully active heterotetramer (A2B2) and inhibited oligomers. Experimental evidence demonstrates that the inhibition of spinach AB‐GAPDH depends on the formation of dimers, tetramers or pentamers of A2B2 modules linked together by C‐terminal extensions of the B subunits that extend from one modular tetramer and occupy two active sites of the adjacent tetramer.
We developed a simple two-dimensional/two-components theoretical model that describes the structure and functionality of a VitE-TPGS system of micelles assuming a hydrophobic inner core and an outer ...hydrated hydrophilic shell. We then conceptually applied the developed methodology to a simple system of VitE-TPGS micelles unloaded and loaded with an active pharmaceutical ingredient, eltrombopag, to verify if the model could reliably monitor the size change of the micelle upon loading. The fit of laboratory Small Angle X-Ray Scattering data against such model allows us to extract absolute values of the micelles size under a spherical shape hypothesis as well as the distribution within the system between components and level of hydration. The intensity scale of the SAXS experimental data needs to be normalized to a reference standard (pure water) to get absolute scattered intensities. The mathematical model which has been developed under a general hypothesis of ellipsoidal micelles, is applied to our experimental data under the simplified spherical assumption, which suitably fits our experimental data.
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Doxorubicin hydrochloride (DX) is one of the most powerful anticancer agents though its clinical use is impaired by severe undesired side effects. DX encapsulation in nanocarrier ...systems has been introduced as a mean to reduce its toxicity. Micelles of the nonionic triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) (PEO-PPO-PEO), are very promising carrier systems. The positive charge of DX confines the drug to the hydrophilic corona region of the micelles. The use of mixed micelles of PEO-PPO-PEO copolymers and a negatively charged bile salt should favour the solubilization of DX in the apolar core region of the micelles.
We studied the DX uptake in the micellar systems formed by sodium cholate (NaC) and the PEO100PPO65PEO100 (F127) copolymer, prepared with different mole ratios (MR = nNaC/nF127) in the range 0 ÷ 1. The systems were characterized by small angle X-ray scattering (SAXS) and dynamic light scattering (DLS); DX encapsulation was followed by steady-state and time-resolved fluorescence spectroscopy.
The successful solubilization of DX in the host micellar systems did not affect their structure, as evidenced by both SAXS and DLS data. In the presence of NaC, DX experiences a more apolar environment as indicated by its characteristic fluorescent behaviour. The almost complete uptake of the drug occurred shortly after the sample preparation; however, time resolved fluorescence revealed a slow partition of DX between corona and core regions of the micelles. DX degradation in the mixed micellar systems was markedly reduced relative to aqueous DX solutions.
A round‐robin study has been carried out to estimate the impact of the human element in small‐angle scattering data analysis. Four corrected datasets were provided to participants ready for analysis. ...All datasets were measured on samples containing spherical scatterers, with two datasets in dilute dispersions and two from powders. Most of the 46 participants correctly identified the number of populations in the dilute dispersions, with half of the population mean entries within 1.5% and half of the population width entries within 40%. Due to the added complexity of the structure factor, far fewer people submitted answers on the powder datasets. For those that did, half of the entries for the means and widths were within 44 and 86%, respectively. This round‐robin experiment highlights several causes for the discrepancies, for which solutions are proposed.
A data‐analysis round robin was performed using four real‐world datasets to quantify the role of the human factor in analysis; the 46 responses show that the analyses by different researchers and laboratories may not be directly comparable, with large reporting inconsistencies in distribution widths and volume fractions. Several underlying causes for these inconsistencies are highlighted that can be addressed by the community.
The association in aqueous mixtures of a thermoresponsive cationic diblock copolymer composed of poly(N-isopropylacrylamide) (PNIPAM) and poly(3-acrylamidopropyl)-trimethylammonium-chloride ...(PAMPTMA(+)) and the oppositely charged bile salt sodium deoxycholate (NaDC) is investigated at different compositions by light and X-ray scattering, calorimetry, and electrophoretic mobility measurements. Clouding reveals aggregation upon heating. The addition of NaDC to the copolymer solution lowers the temperature of the transition and increases its cooperativity. At high temperature and low NaDC fractions, mixed aggregates with a dehydrated PNIPAM-rich interior and a PAMPTMA(+)-rich shell partially neutralized by DC– anions are formed. At high NaDC fractions, the aggregates present internal regularly spaced segregated nanoregions of dehydrated PNIPAM and PAMPTMA(+)/DC– (microphase separation). The results suggest that the mixed aggregates have appealing composition-controlled thermoresponse. The system phase separates at body temperature and the highest NaDC fractions investigated, meaning in conditions accomplished when the use of the polymer as a bile salt sequestrant is hypothesized.
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•Temperature dependent association of a cationic poly(N-isopropylacrylamide) block copolymer and a bile salt was studied.•Phase transition temperature of block copolymer lowered by adding bile salt.•At high temperature and low bile salt fractions, mixed aggregates with dehydrated PNIPAM-rich interior were formed.•At high temperature and high bile salt fractions, the mixed aggregates presented regularly spaced segregated nanoregions.•The promising application of the block copolymer as a bile salt sequestrant could be hypothesized at body conditions.
Condensation of DNA helices into hexagonally packed bundles and toroids represents an intriguing example of functional organization of biological macromolecules at the nanoscale. The condensation ...models are based on the unique polyelectrolyte features of DNA, however here we could reproduce a DNA‐like condensation with supramolecular helices of small chiral molecules, thereby demonstrating that it is a more general phenomenon. We show that the bile salt sodium deoxycholate can form supramolecular helices upon interaction with oppositely charged polyelectrolytes of homopolymer or block copolymers. At higher order, a controlled hexagonal packing of the helices into DNA‐like bundles and toroids could be accomplished. The results disclose unknown similarities between covalent and supramolecular non‐covalent helical polyelectrolytes, which inspire visionary ideas of constructing supramolecular versions of biological macromolecules. As drug nanocarriers the polymer–bile salt superstructures would get advantage of a complex chirality at molecular and supramolecular levels, whose effect on the nanocarrier assisted drug efficiency is a still unexplored fascinating issue.
Chiral bile salt biosurfactant with low molecular weight can self‐assemble into supramolecular helices by interaction with oppositely charged polymers. A controlled condensation of the supramolecular helices into DNA‐like hexagonally packed bundles and toroids can be achieved when using block copolymers of opposite charge and increasing the bile salt content.
In land plants and algae, the Calvin–Benson (CB) cycle takes place in the chloroplast, a specialized organelle in which photosynthesis occurs. Thioredoxins (TRXs) are small ubiquitous proteins, known ...to harmonize the two stages of photosynthesis through a thiol-based mechanism. Among the 11 enzymes of the CB cycle, the TRX target phosphoribulokinase (PRK) has yet to be characterized at the atomic scale. To accomplish this goal, we determined the crystal structures of PRK from two model species: the green alga Chlamydomonas reinhardtii (CrPRK) and the land plant Arabidopsis thaliana (AtPRK). PRK is an elongated homodimer characterized by a large central β-sheet of 18 strands, extending between two catalytic sites positioned at its edges. The electrostatic surface potential of the catalytic cavity has both a positive region suitable for binding the phosphate groups of substrates and an exposed negative region to attract positively charged TRX-f. In the catalytic cavity, the regulatory cysteines are 13 Å apart and connected by a flexible region exclusive to photosynthetic eukaryotes—the clamp loop—which is believed to be essential for oxidation-induced structural rearrangements. Structural comparisons with prokaryotic and evolutionarily older PRKs revealed that both AtPRK and CrPRK have a strongly reduced dimer interface and an increased number of random-coiled regions, suggesting that a general loss in structural rigidity correlates with gains in TRX sensitivity during the molecular evolution of PRKs in eukaryotes.
Condensation of DNA helices into hexagonally packed bundles and toroids represents an intriguing example of functional organization of biological macromolecules at the nanoscale. The condensation ...models are based on the unique polyelectrolyte features of DNA, however here we could reproduce a DNA‐like condensation with supramolecular helices of small chiral molecules, thereby demonstrating that it is a more general phenomenon. We show that the bile salt sodium deoxycholate can form supramolecular helices upon interaction with oppositely charged polyelectrolytes of homopolymer or block copolymers. At higher order, a controlled hexagonal packing of the helices into DNA‐like bundles and toroids could be accomplished. The results disclose unknown similarities between covalent and supramolecular non‐covalent helical polyelectrolytes, which inspire visionary ideas of constructing supramolecular versions of biological macromolecules. As drug nanocarriers the polymer–bile salt superstructures would get advantage of a complex chirality at molecular and supramolecular levels, whose effect on the nanocarrier assisted drug efficiency is a still unexplored fascinating issue.
Chiral bile salt biosurfactant with low molecular weight can self‐assemble into supramolecular helices by interaction with oppositely charged polymers. A controlled condensation of the supramolecular helices into DNA‐like hexagonally packed bundles and toroids can be achieved when using block copolymers of opposite charge and increasing the bile salt content.
In this work, the diblock copolymer methoxy-poly(ethylene glycol)-block-poly(ε-caprolactone) (MPEG–b-PCL) was synthesized with a block composition that allows this polymer in aqueous media to ...possess both an upper critical solution temperature (UCST) and a lower critical solution temperature (LCST) over a limited temperature interval. The value of the UCST, associated with crystallization of the PCL-block, depended on heating (H) or cooling (C) of the sample and was found to be CPUCST H = 32 °C and CPUCST C = 23 °C, respectively. The LCST was not affected by the heating or cooling scans; assumed a value of 52 °C (CPLCST H = CPLCST C). At intermediate temperatures (e.g., 45 °C), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM) showed that the solution consisted of a large population of spherical core–shell particles and some self-assembled rodlike objects. At low temperatures (below 32 °C), differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) in combination with SAXS disclosed the formation of crystals with a cylindrical core–shell structure. Cryo-TEM supported a thread-like appearance of the self-assembled polymer chains. At temperatures above 52 °C, incipient phase separation took place and large aggregation complexes of amorphous morphology were formed. This work provides insight into the intricate interplay between UCST and LCST and the type of structures formed at these conditions in aqueous solutions of MPEG–b-PCL diblock copolymers.
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